a thesisa thesis - iaea

Ain Shams University Woman's the College Arts, Science &Education

IMPROVING CITRIC ACID PRODUCTION FROM SOME CARBOHYDRATES BY-PORODUCTS USING IRRADIATED

ASPERGILLUS NIGER

A thesisA thesisA thesisA thesis

Submitted for Ph.D Degree of science

in microbiology

to

the Botany Department

Women's College for Arts, Science & Education

Ain Shams University ,Cairo, Egypt

ByByByBy

Souzy Sobhy Farag B.Sc. of Science (Botany) Fac. of Sci, Ain Shames University 1982

M.Sc. of Sciences (Microbiology) Fac. of Sci, Zagazig University 2003

2011

Ain Shams University Woman's the College Arts, Science &Education

AcknowledgmentAcknowledgmentAcknowledgmentAcknowledgment

First, thanks for God for helping me to do this work.

I would like to express my deep gratitude to Prof. Dr. Fatma

Abd-El-Wahab Helimesh, professor of Microbiology, Women's

College, Ain Shams University Cairo, Egypt, for her kind supervising

this work and continuous valuable guidance.

My deep gratitude and sincerely thanks to Prof. Dr. Nahed Ali

Younis, Professor of Microbiology, Plant Research Department,

Nuclear Research Center, Atomic Energy Authority, for supervising

this work and her great efforts during the study, my sincers and cordial

appreciation.

Also I'm greatly indebted to thank with deep appreciation Dr.

Sherif Moussa Husseiny, Assistant Prof. of Microbiology Women's

College, Ain Shams University , Cairo, Egypt for suggesting the point

of the study, his kind supervision, and continuous guidance through

out the course of this work.

My sincere thanks are extended to Dr. Hany Wahib Botors,

Lecturer of Microbiology, Pant Research Department, Nuclear

research Center, for his cooperation during the study, My thanks to

Dr. Hanan Abd Alla Abdel Menem Lecturer of Biochemistry, Biological Application Department Nuclear Research Center for her

efforts in explanation PCR data also my thanks to all members of

Microbiology Unit for their useful help.

Finally, my sincere thanks are to the chairman of Nuclear Research

Center, and also to the Head of Plant Research Department – in

addition to Head of Plant and Microbiology Women's College, Ain

Shams University, Cairo, Egypt.

ABSTRACT

Twenty strains of A. niger were isolated from different sources,

screened for their capacity to produce citric acid. All the isolated strains were

able to produce citric acid in different quantities at different time intervals i.e.

4, 8 and 12 days on indicator medium. The best incubation period for

production for all isolates was 12 days. The most potent strains for production

were A1, A4 & A5, while A8, A16, A18 & A19 recorded weak production on that

medium. Citric acid productivity were obtained by all strains when using

different concentrations of four carbohydrate by-products (maize straw,

potato peel wastes, sugar beet pulp and molasses) when each used alone

without any additions after 12 days incubation and the production enhanced

when the fermentation medium amended with the same concentrations of the

mentioned substrates. Type and concentration of carbohydrate by-product

affect the production of citric acid by A. niger strains under the study.

Increasing substrate concentration led to increase in production, the best

concentration for production was 25% for all carbohydrate by-products. As

recorded with indicator medium, A1, A4 & A5 are also the most potent strains

for production when growing on the four carbohydrate by-products

supplemented to the basal medium, while A8, A6, A18 & A19 recorded the weak

production with the carbohydrate by-products used.

Production of the parental isolates A1, A4 & A5 on indicator medium

were: 0.96, 0.95 and 0.99 (mg/ml) respectively after 12 days incubation, while

maximum production by the obtaining resulting isolates (Treated by uv

irradiation) were: 1.78, 1.70 and 1.73 (mg/ml) from A4T2 (5min.), A4T1

(10min.) and A1T1 (5min.), respectively. Production of mentioned parental

isolates on basal medium with 25% maize straw were: 0.73, 0.70 and 0.85

respectively at the same incubation period, while maximum yield from the

obtained ones (Treated) were: 1.50, 1.45 (mg/ml) by A4T2 (5min.) and A4T1

(10min.), respectively. Production of parental selected isolates on basal

medium with 25% potato peel wastes: 0.94, 0.91 & 0.95 (mg/ml) respectively,

while maximum yield by the treated ones at the same medium were 1.85, 1.84

& 1.78 (mg/ml) by A1T1 (5min.), A1T2 (5min.) and A1T3 (5min.), respectively.

On basal medium with 25% sugar beet pulp parental production were 1.60,

1.50 and 1.51 (mg/ml), respectively after 12 days incubation, while maximum

yield by the derivatives at the same medium reached to 3.16 and 2.82 (mg/ml)

by: A1T1 (5min.) and A1T5 (min.). Parental isolates A1, A4 & A5 growing on

basal medium with 25% molasses recorded: 1.22, 1.04 and 1.20 (mg/ml),

respectively, while the yield reached from (5.00-5.62 mg/ml) for 17 obtained

treated ones from parental A1 after exposure time (2, 5, 10, 15 and 20 min.) to

uv irradiation with increases 4 folds approximately. Parental weak isolates

(A8, A16, A18 & A19) recorded 0.74, 0.70, 0.69 and 0.60 mg/ml, respectively on

indicator medium. Increase 8, 7, 6 & 5 folds by the treated ones. Maximum

production was 5.35 mg/ml recorded by A19T1 (10 min). Maximum production

obtained by γγγγ-rays in the study ranged from 2.00 – 2.40 mg/ml, increase 2 fold

on indicator medium and basal medium amended with the carbohydrate by-

products for some treated ones.

A1 & AT1 (5 min) also A19 & A19T1 (10 min) were chosen for PCR

study.RAPD-PCR analysis revealed that five positive unique markers

amplified by certain primers (mentioned in the study) identified the mutant

A1T1 (5 min), while in the wild isolate A1 there were nine unique markers

amplified by certain primers. Sixteen positive unique markers amplified and

identified the mutant A19T1 10 min, while in the wild isolate A19 twelve unique

markers were amplified by mentioned primers. The genetic similarity

between two genotypes A1 and its mutant A1T1 (5 min) is 81% and variation is

19% while the genetic similarity between A19 and its mutant A19T1 (10 min) is

73.6% and variation 26.4%.

In protein profile (SDS-PAGE) twenty two bands were obtained in A1

ranged from 243.2 to 30.1 KDa while its mutant A1T1 (10 min) have fifteen

bands ranged from 235.7 to 31.9 KDa. Also A. niger A19 showed twelve bands

ranged from 91.12 to 24.78 KDa while after uv irradiation 10 min mutant

obtained isolate A19T1 (10 min) showed sixteen bands ranged from 100.74 to

21.15 KDa.

CONTENTS Page

List of figures………...……………………………..…… --

List of tables ………..……………………………..…….. --

List of plates………..……………………………..……... --

ACKNOWLEDGEMENT

ABSTRACT …….………………………………………. I

INTRODUCTION………………………………………. 1

AIM OF THE WORK …………………………………... 4

REVIEW OF LITERATURE …………………………… 5

Natural citric acid ……………………………………… 5

Citric acid cycle …………………………………………. 5

Manufacture citric acid ....................................................... 6

Industrial importance of citric acid ..................................... 6

Biosynthesis of citric acid ................................................... 8

The possibility of utilizing carbohydrate by-products for

citric acid production by A. niger ................................... 11

Isolation of mutants of A. niger for hyperproduction of citric

acid through uv light irradiation……….....................…….. 14


acid through γ rays ..................................................... 17

MATERIALS AND METHODS .................................... 21

Microorganisms (parental strains) ...................................... 21

Inoculum preparation ……………………………………. 21

Carbohydrate by-products ................................................. 22

Cultivation .......................................................................... 22

Exposure of selected parental strains of Aspergillus niger

to uv light irradiation and γ-rays: .................................... 22

Isolation of obtained strains (treated isolates)..................... 23

Assay of citric acid ............................................................ 23

Random Amplified Polymorphic DNA Polymerase Chain

Reaction (RAPD-PCR) ....................................................... 24

Sodium dodecyl sulfate (SDS) - Polyaciylamide gel.......... 27

Protein electrophoresis........................................................ 31

RESULTS …………………………………….…………. 35

Part I: Selection the most potent strains of A. niger growing on

a modified Czapek's agar medium supplemented by

different carbohydrate by-products for citric acid

production………………………………………. 34

1. Production of citric acid by Aspergillus niger strains on

indicator medium............................................................. 35


maize straw after 12 days incubation.............................. 40

3. Production of citric acid by Aspergillus niger isolates on

potato solid wastes (peels) after 12 days incubation........ 44


sugar beet pulp after 12 days incubation........................... 44


molasses after 12 days incubation..................................... 48

Part II: Yield of citric acid by treated (potent & weak)

strains of Aspergillus niger that growing on

different carbohydrate by-products using uv light

irradiation & γ-rays ............................................... .. 62

1. Influence of uv light irradiation & γ -rays on the most

potent strains A. niger for citric acid production............. 63

A. U.V light irradiation..................................................... 63

B. γ-rays ........................................................................... 75

2. Influence of uv light irradiation on weak isolates of

A. niger of citric acid production..................................... 91

Part III: RAPD-PCR amplification and protein

electrophoresis for A. niger isolates.......................100

DISCUSSION ……………………………………………. 126

SUMMARY……………………………………………… 136

CONCLUSION …….……………………………………. 144

RECOMMENDATION ………………………………... 145

REFERENCES …………………………………………. 146

ARABIC SUMMARY…………………………………….

List of Figures

Fig. Subject Page

1 Standard curve citric acid. 24

2 Production of citric acid (mg/ml) by different

strains of Aspergillus niger using indicator

medium after 12 days incubation

38

3 Maximum Citric acid production by A. niger

isolates (A1, A4 & A5) on indicator medium at

different time intervals

39

4 Weakest production of citric acid by four isolates

(A8, A16, A18 & A19) on indicator medium at


39

5 Citric acid production (mg/ml) by different

isolates of A. niger growing on 25% maize straw

after 12 days incubation

43


isolates of A. niger growing on basal medium

amended with 25% maize straw after 12 days

incubation

43


isolates of A. niger growing on 25% of potato

solid wastes after 12 days incubation

47



amended with 25% of potato solid wastes after 12

days incubation

47


isolates of A. niger growing on 25% of sugar beet

pulp after 12 days incubation

51


isolates of A. niger growing on basal liquid

medium amended with 25% of sugar beet pulp


51


isolates of A. niger growing on 25% of molasses


54

List of Figures (Cont.)

Fig. Subject Page



medium amended with 25% of molasses after

12 days incubation

54

13 Citric acid production (mg/ml) by most potent

isolates on basal medium amended with

different concentration of maize straw

56

14 Citric acid production (mg/ml) by weak


different concentration of maize straw

56



different concentration of potato solid wastes

57



different concentration of potato solid wastes

57



different concentration of sugar beet pulp

58



different concentration of sugar beet pulp

58



different concentration of molasses

59



different concentration of molasses

59


Fig. Subject Page

21 Citric acid production (mg/ml) of selected

parental isolate A1 and its obtained isolates

after uv irradiation after 12 days incubation on

indicator medium

68




indicator medium

68




indicator medium

68




basal medium amended with 25% maize straw

70





70





70


Fig. Subject Page




basal medium amended with 25% potato solid

wastes

72





wastes

72





wastes

72




basal medium amended with 25% sugar beet

pulp

74





pulp

74





pulp.

74


Fig. Subject Page




basal medium amended with 25% molasses

77





77





77



after gamma rays irradiation after 12 days

incubation on indicator liquid medium

81



after gamma rays irradiation rays after 12 days


81





81




incubation on basal medium amended with

25% maize straw

83


Fig. Subject Page





25% maize straw

83





25% maize straw

83





25% potato solid wastes

86






86






86





25% sugar beet pulp

88

List of Figures (Cont.) Fig. Subject Page





25% sugar beet pulp

88





25% sugar beet pulp

88





25% molasses

90





25% molasses

90





25% molasses

90

51 Citric acid production (mg/ml) of parental

weak isolate A8 and its obtained isolates after

uv irradiation after 12 days incubation on

indicator medium

96




indicator medium

96

List of Figures (Cont.) Fig. Subject Page




indicator medium

96




indicator medium

97

55 Citric acid production (mg/ml) by A19 (T1 uv

10) of A. niger on different carbohydrate by

products after 12 days incubation.

97

56 Substrate consumed (%) by A19 (T1 uv 10) of

A. niger on different carbohydrate by products


97

57 RAPD banding patterns amplified for A. niger

(A1) and its obtained isolate(A1T15min) using

primers(OPO-14&OPO-02) M (DNA marker)

= 5Kb ladder

104


(A1) and its obtained isolate (A1T15 min) using

primers(OPB-10&OPB-08) M (DNA marker)

= 1Kb ladder

104


(A1) and its obtained isolate (A1 T1 5min)

using primer (OPA-05)M (DNA marker) =

1Kb ladder.

105


(A1) and its obtained isolate (A1T1 5min) using

primers (OPA-04, OPA-15 & OPA-18) M

(DNA marker) = 1Kb ladder

105


Fig. Subject Page


(A1) & its obtained isolate (A1 T1 5min) using

primers (OPC-14 &OPB-05)M (DNA marker)

= 1kb ladder.

106


(A19) and its obtained isolate (A19 T1 10 min)

using primer OPG-0.5 M (DNA marker) = 1kb

ladder

108



using primer OPH-15 M (DNA marker) = 100

base pairs ladder.

109



using primer OPC-10 M (DNA marker) = 1kb

ladder.

109


(A19) and its obtained isolated (A19 T1 10min)

using primer OPC-20 M (DNA marker) = 1

kb ladder

110

66 RAPD banding patterns amplified for A niger

(A19) and its obtained isolate (A19 T1 10 min)

using primer OPA-20 M (DNA marker) = 1kb

110



using primer OPO-04 M (DNA marker) = 1Kb

111



using primer OPB-15 M (DNA marker) = 1kb

ladder.

111


Fig. Subject Page

69 RAPA banding patters amplifying for A. niger

(A19) and its isolate (A19 T1 10min) using

primer OPB-05 M (DNA marker) = 1kb

ladders

112


(A19) & obtained isolate (A19 T1 10min) using

primer OPB-07 M (DNA marker) = 100 base

ladder

112



using OPB-06 M (DNA marker) = 1kb ladder

113

72 Marker Lane Profile 121

73 Lane A1 T1 Profile 122

74 Lane A1 Profile 123

75 Lane A19 T1 Profile 124

76 Lane A19 Profile 125

List of Tables

Table Subject Page

1 Source of isolation for twenty strains of A.

niger

36

2 Diameter of clear zone (mm) and production

of citric acid (mg/ml) by different strains of

Aspergillus niger using indicator medium at


37


isolates of A. niger grown on different

concentrations of maize straw after 12 days

incubation

41



amended with different concentrations of

maize straw after 12 days incubation

42


isolates of A. niger grown on different

concentrations of potato solid wastes after 12

days incubation

45


isolates of A. niger grown on basal medium

amended with different concentrations of

potato solid wastes after 12 days incubation

46


isolates of A. niger growing on different

concentrations of sugar beet pulp after 12 days

incubation

49



medium amended with different concentra-

tions of sugar beet pulp after 12 days

incubation

50

List of Tables (Cont.)

Table Subject Page


isolates of A. niger growing on different

concentrations of molasses after 12 days

incubation

52



medium amended with different concentra-

tions of molasses after 12 days incubation

53

11 Number of colonyforming units of selected

isolates of A. niger (A1, A4, & A5) on indicator

medium after exposure to uv irradiation

64

12 Screening of treated isolates obtained after uv

irradiation of selected A. niger isolates (A1, A4

and A5) for production of citric acid on

indicator medium in terms of diameters of

clearing zone (mm) at different time intervals

65


parental isolates of A. niger (A1, A4 and A5)

and obtained isolates (treated) after uv

irradiation on indicator medium

67




irradiation growing on basal liquid medium

amended with 25% maize straw

69


Table Subject Page





amended with 25% potato solid wastes

71





amended with 25% sugar beet pulp

73





amended with 25% molasses

76

18 Number of colony-forming units of selected

isolates of A. niger (A1, A4 and A5) on

indicator medium after exposure to gamma

rays

78

19 Screening of treated isolates obtained after

gamma rays irradiation of selected A. niger

isolates (A1, A4 and A5) for production of

citric acid and obtained isolates on indicator

medium in terms of diameter of clearing zone

(mm) at different time intervals

79



and obtained isolates (treated) after gamma

rays irradiation on indicator liquid medium

80


Table Subject Page

21 Citric acids production (mg/ml) of selected



rays irradiation growing on basal liquid

medium amended with 25% maize straw

82





medium amended with 25% potato solid

wastes

85





medium amended with 25% sugar beet pulp

87





medium amended with 25% molasses

89

25 Number of colony-forming units of weak

isolates of A. niger (I8, I16, I18 and I19) on

indicator medium after uv irradiation

93

26 Screening of treated isolates obtained after uv

irradiation of parental weak isolates of A.

niger (A8, A16, A18 and A19) for production of

citric acid on indicator medium in terms of

diameter of clearing zone (mm) at different

time intervals

94


Table Subject Page

27 Citric acid production by parental weak

isolates (A8, A16, A18 and A19) of A. niger and

obtained isolates after uv irradionlion on

indicator medium at different time intervals

95

28 Citric acid production (mg/ml) by A19 (T1 uv

10) of A. niger on different carbohydrate by

products after 12 days incubation

98

29 Number of amplified DNA bands scored for

the wild strain A. niger A1 (potent isolate) and

its obtained isolate (A1 T1 5min) by uv

irradiation.

102

30 Positive and negative unique RAPD markers

and their molecular weight of the two strains

of A. niger (A1 & A1 T1 5 min).

103

31 Number of amplified DNA bands scored for

the wild strain of A. niger A19 and its obtained

isolate (A19T1 10 min) by uv irradiation.

107

32 Positive and negative unique RAPD markers

and their molecular weight for wild A. niger

A19 (weak isolate) and its obtained isolate

(A19T1 10min) after treatment with uv

irradiation.

115

33 RAPD based genetic similarity (GS) between

genotypes of A. niger: (A1 & A1 T1 5 min)-

(A19 & A19T1 10min).

116

34 SDS-PAGE for (A1 &A1 T1 5 min and A19 &

A19 T1 10 min.)

118

List of Plates

Plates Subject Page

1,2 & 3 Most potent isolates of A. niger for citric

acid production on indicator medium after

12 days incubation

60

4, 5, 6 & 7 Weak isolates of A. niger for citric acid

production on indicator medium after 12

days incubation

61

7 Wild (Parental isolate) of A. niger (I 19) on

indicator medium after 12 days incubation

99

8 Derivative isolate of A. niger (I 19) after

exposure to uv light irradiation (10 minutes)

after 6 days incubation on indicator medium

(A19 T1).

99

- 1 -

Introduction

Citric acid (C6 H5 07) i.e., 3 hydroxy 1, 3, 5 propane

tricarboxylic acid is ubiquitous in nature and exists as an

intermediate in citric acid cycle when carbohydrates are oxidized

to carbon dioxide. It is solid at room temperature, melts at 153°C

and decomposes at higher temperature into other products

(Rojoka et al., 1998). It is responsible for tart taste of various

fruits in which occurs i.e. lemon, lime, figs, oranges, pineapples,

pears and gooseberries (Francis, 2000). It is non toxic and easily

oxidized in the human body. Because of its high solubility,

palatability and low toxicity, it can be used in food, biochemical

and pharmaceutical industries, these uses have placed greater

stress on increased citric acid production and search for more

efficient fermentation process. The world wide demand of citric

acid about 6.0 x105 tons per year and is bound to increases day by

day (Ali et al., 2001).

In 2004, the world wide production of citric acid was

approximately 1.4 million tons according to the business

communications Co. (BCC)'s recent studies of fermentation

(Soccol et al., 2006). Moreover due to its large application and

low price, the citric acid consumption is expected to grow

significantly until 2009, and this raises the need for industries to

search for new technological alternative and for cost reduction in

citric acid production (Vandenberghe et al.,2004) citric acid

(CA) has a variety of applications, 70% of it used in food and

- 2 -

beverage industries, 12% in the pharmaceutical industry and 18%

in other industries (Soccol et al., 2003 Pandey et al., 2001). A

potentially promising economical and efficient source for

producing citric acid is to use the fungus A. niger due to its

higher capacity to accumulate acid when compared to other

organisms (Adham, 2002; Pazauki et al., 2000, Pera and

Callieri, 1999; Maddox and Brooks, 1998 and Fiedurk et al.,

1996 and Yokoya, 1992). It has been found to be an efficient

producer of citric acid when cultured media comprised of residues

and extracts such as sugar and molasses (Mehyar et al.,2005;

Bayraktar and Mehmetaglu, 2000; El-Holi, 1999; Roukas,

1998; 1991; Gutierrez Rojas, 1995; Gupta and Sharma, 1994

and Khare et al.,1994).

Possibilities have been done for using some by-products

and agroindustrial residues in citric acid production by solid state

fermentation, on the other hand, solving in serious environmental

problems (Rodrigues et al., 2010; Darani and Zoghi, 2008;

Soccol and Vandenberghe, 2003; Prado, 2002; Pintado et

al.,1998; Soccol, 1996, and Kolicheski 1995). Citric acid

production by A. niger depends on several specific nutritional and

environmental conditions as well as the particular strain of

microorganisms. It is a primary metabolite of Kreb's cycle

production from reacting pyruvate with acetyl CoA which is

catalyzed by pyruvate carboxylase (Hagedorn and Kaphammer

1994; Stanburry and Whitaker 1984).

- 3 -

Mutations are abrupt and so are the hereditary

modifications in the genetic material. The organism containing

the DNA are not static molecules and their bases are frequently

exposed to natural or artificial agents can cause modification in

their structure or in chemical composition (Griffiths et al., 2006

and Zaha 2003). The increase in citric acid productivity has been

achieved using mutation and strain selection. Mutation strains

with certain characteristics such as enhanced citric acid

production and increased fermentation rate have been previously

selected after submitting the genetic material to physical or

chemical mutagenic agents (Lotfy et al., 2007, Griffiths et

al.,2006, Ikram-Ul Hag et al.,2001 and Rohr et al.,1983).

The most frequently used method is induction by UV

irradiation that can randomly provide a strain with a higher

capacity of citric acid production when compared to the control

strain (Griffiths et al.,2006 and Zaha, 2003). γ-rays as

mutagenic agent used for improve citric production by A. niger

(Alani et al.,2007) investigated optimization of citric acid

production from a new strain and mutant of A. niger after γ-ray

irradiation using solid state fermentation. In addition Parvez et

al.,1998 obtained mutant strain of A. niger for citric acid

production after γ-irradiation.

- 5 -

Aim of the work

Accordingly, this work was undertaken to through light upon

the following aspects:

1. Isolation and screening of different strains of A.niger for their

ability to grow and produce citric acid.

2. Selection of the most potent and weak isolates for citric acid

production.

3. Utilization of different carbohydrate by-products as substrates

for citric acid production by isolates of A.niger.

4. Studying of the influence of mutagenic agents (uv light

irradiation and γ-rays) on the most potent and weak isolates.

5. Quantitative estimation of citric acid production by irradiated

strains that allows to grow on carbohydrate by-products as

carbon and energy source.

6. Studying of some molecular studies on irradiated strains

- RAPD-PCR

- protein profile.

- 7 -

Review of Literature

Natural citric acid:

Bouchard & Merritt, 1979 reported that the total

circulating citric acid in the serum of man is approximately

1mg/kg of body weight. Beli & Odian, 2000 and Francis, 2000

showed that citric acid is found naturally in almost all living

things both plant and animal. It is predominant acid in substantial

quantity' in citrus fruits (oranges, lemons, limes, etc..,), in berries

(strawberries, raspberries) and in pineapples. Citric acid is also

predominat acid in many vegetables such as potatoes, tomatoes,

asparagus, turnips and peas but in lower concentrations. Nill,

2002 observed that some plants naturally release citric acid from

their roots into the surrounding soil, in order to "bind" aluminum

ions that are present in some soils, such aluminum which slows

plant growth and decrease crop yields is present to a certain

degree (which causes at least some crop yield reduction). CH2 COOH |

HO C – COOH |

CH2 COOH

Citric acid (3-hydroxyl 1, 3, 5 propane-tricarboxylic acid)

Citric acid cycle:

A system of enzymatic reactions in which acetyl residues

are oxidized to carbon dioxide and hydrogen atoms. It is also

- 8 -

known as the tricarboxylic acid cycle (TCA cycle) or Kerbs cycle

occurs in almost all aerobic (air requiring) organisms (Nill, 2002).

Manufacture of citric acid:

Francis, 2000 reported that citric acid is manufactured by

fermentation, a natural process using living organisms. The acid is

recovered in pure crystalline form either as the anhydrous or

monohydrate crystal depending on the temperature or

crystallization. The transition temperature is 36.6°C.

Crystallization above this temperature produces an anhydrous

product while the monohydrate forms at lower temperatures. The

anhydrous form is preferred for its physical stability and its more

widely available commercial form.

Industrial importance of citric acid:

Higgins et al., 1985 reported that citric acid has a pleasant

acid taste and is soluble in water, it is widely used in food

industry, in pharmaceuticals and cosmetics, the esters of the acid

are used in the plastic industry and as a metal chelator, citric acid

finds application in metal purification and as a readily

biodegradable ingredient of detergents. Kishore & Smis, 1993;

Pallares et al., 1995 mentioned that citric acid is widely used in

the food, pharmaceutical and chemical industries due to its

distinctive properties as an acidulent, flavoring agent, antioxidant

and high solubility.

- 9 -

Francis, 2000 mentioned to the broad spectrum for using

citric acid and its salts in food and beverage products. i.e.,

beverages, gelation desserts, backed goods, jellies, jams,

preserves, candies, fruits and vegetables, dairy products, meats,

seafood and fats & oils. Soccol et al., 2003 pointed that buffering

properties of citrates are used to control pH in household cleaners

and in pharmaceuticals. Luciana et al., 2004. showed that the

food industry is the consumer of citric acid using almost 70% of

the total production followed by about 12% by the pharmaceutical

industry and 18% for other applications.

Xie & West, 2006 said that the estimated world production

of citric acid was reported as 1.000.000 tons/year and the world

market demand is increasing day by day. Lotfy et al., 2007

concluded that citric as is one of the of the few bulk chemicals

produced by fermentation. It has a broad use in the household, in

the preparation of numerous industrial products and in many

industrial areas such as the food, pharmaceutical, and chemical

industries, and as a cleaning agent.

Biosynthesis of citric acid:

Different microorganisms had the ability to synthesize

citric acid. Among these microorganisms that considered as active

producers of citric acid: Aspergillus niger, penicillium lactum,

Mucor pyri formis, Tricoderma viride, (Klrimura et al., 1990).

- 10 -

The variety of yeasts known to produce citric acid from

various sources are species of Candida, Saccharomyces,

Yarrowia, (Good et al., 1985; El-Sayed 1986; Kishore & Smis,

1993 and Crolla & Kennedy, 2001). Arzumanov et al., 2000

reported that various strains of fungi which have been found to

accumulate citric acid in their culture media include strains of A.

niger, A. awamori, Penicillium restrictum, Trichloderma viride,

Mucor Piriformis and Yarrowia Lipolytica, but A. niger remained

the organism of choice for the production of citric acid.

Mourya and Jauhri, 2000 demonstrated that A. niger has

been the organism of choice due to its ease of handling, ability to

use a variety of cheap raw materials and high yield of citric acid,

also they screened seventeen strains of A. niger for their capacity

to produce citric acid using starch hydrolysate as a substrate, the

most efficient strain TICC-605 was selected for further

improvement in citric acid content by

- 11 -

The citric acid cycle. Two carbon atoms enter the cycle in the form of acetyl-CoA and are joined to oxaoacetate to form citrate (Step 1). During the series of reaction that then occur, two molecules of carbon dioxide are formed (Steps 5 and 6), and four pairs to of hydrogens are produced and carried to the respiratory chain (Steps 4, 6, 8 and 10). At the end to the cycle a molecule of oxaoacetate is formed, ready for another turn of the cycle (Step 10).

- 12 -

mutation. Hang and Woodams, 2001 studied enzymatic

enhancement of citric acid production by A. niger from corncobs.

Ishaq et al., 2002 studied time course profile of citric acid

fermentation by A. niger and its kinetic relations and found that

mutant strain of A. niger GCB47 was a faster growing organism

and has the ability to hyper produce citric acid. Asad et al., 2003

evaluated phosphate limitation for enhanced citric acid

fermentation using A. niger mutant uv- µ9 on semi-pilot scale.

Kurbanoglu, 2004 studied the potential use of ram horn

hydrolysate (RHH) as a supplement for improvement of citric

acid production by A. niger NRRL 330. Prado, et al., 2005

worked to study the relation between citric acid production and

respiration of A. niger LPB 21 in solid state fermentation of

cassava baggasse, the experiment was carried out in horizontal

drum bioreactor coupled with gas chromatography system. Kim

et al., 2006 studied nutrient optimization for the production of

citric acid by A. niger NRRL 567 grown on peat moss enriched

with glucose.

Lotfy et al., 2007 demonstrated to citric acid production by

a noval A. niger isolate and studied the optimization of process

parameters through statistical experimental designs. Barrington

and Woo, 2008 found that maximum citric acid production in

- 13 -

optimized condition by central composite design (CCD)

represented about a 2.7 fold increase compared to that obtained

from control before optimization when A. niger NRRL 567 grown

in peat moss. Rodrigues et al., 2010 aimed to optimize the

physical-chemical conditions of citric acid through a careful strain

selection of A. niger by solid state fermentation, the best

production was reached at 65%. moisture, 30oC and pH 5.5.

The possibility of utilizing carbohydrate by-products for citric

acid production by A. niger:

Considerable interest has been developed in using

agricultural wastes as substrates for citric acid production

including pineapple wastes (Tran & Mitchell 1995 and Tran et

al., 1998), grape pomace (Hang & Woodams 1995), apple

pomace (Fatemi & Shojaosadati, 1999; Shojaosadati &

Babaripour, 2002), corn cobs (Hang and Woodams 2001), kiwi

fruit peel (Kumar et al., 2003), cassava bagasse (Vandenberghe

et al., 2004 & Flavera et al., 2005).

Xie & West, 2006 screened seven citric acid producing A.

niger for their ability to synthesize citric acid on untreated and

autoclaved wet corn distillers grains by solid-state fermentation.

The most effective citric acid producing strain of A. niger was

ATCC 9142 on the untreated or autoclaved grains. The

- 14 -

autoclaved grains supported less citric acid production by the

majority of strains screened.

Different agro-industrial residues have been investigated as a

substrate for citric acid production. El-Batal et al., 1995 studied

five strains of Aspergillus niger (EMCC 102, EMCC 104, EMCC

111, EMCC 132 and EMCC 147) were used for citric acid

production at different incubation periods using different cheap

carbohydrate substrates, such as beet, cane, citrus molasses and

milk whey. A. niger EMCC111 was found to be the most potent

strain for citric acid production from beet molasses after 11 days

of incubation at 30oC.

Ikram-ul-Hag et al., 2003, studied production of citric

acid from raw starch by Aspergillus niger. Shake flask and semi-

solid culture methods were compared using A. niger GCB-47

(parental strain) and GCMC-7 (mutant strain). In shaking culture

with 150g/L soluble starch as a carbon source, the mutant strain

GCMC-7 produced 69.5 g/L citric acid, which was 1.48 fold

greater than the parental strain GCB-47. Direct production of

citric acid from corn and potato starch was examined using semi-

solid culture. It was observed that the mutant strain was a faster

growing organism. The mutant strain GCMC-7 produced 71.4 and

92.9 g/L citric acid approximately 1.12 and 1.44 times as much as

the parental strain GCB-47 from 200 g/L corn and potato starch,

- 15 -

respectively. The finding suggest that GCMC-7 possesses

enhanced ability for sugar metabolism and citric acid production.

Asad et al., 2003 evaluated twenty five strains of A. niger,

isolated from different soil samples for citric acid production

using cane-molasses medium. Vandenberghe et al., 2004

evaluated three different agroindustrial wastes, sugar cane

bagasse, coffee husk and cassava bagasse for their efficiency in

production of citric acid by a culture of Aspergillus niger under

solid-state fermentation. Cassava bagasse supported best fungal

growth giving the highest yield of citric acid among the tested

substrates. Results showed that the fungal strain had good

adaptation to the cassava bagasse substrate and increased the

protein content in the fermented matter.

Lotfy et al., 2007 studied reduction of the fermentation

medium cost, corn steep liquor and calcium phosphate pre-treated

beet molasses were successfully used as a subsituents of nitrogen

and carbon sources in the growth medium of A.niger UMIP 2564

respectively. These medium substitutions resulted in citric acid

fermentation culture with a product yield of 74.56%.


acid through uv light irradiation:

Hamissa et al., 1992 used different doses of uv irradiation

to obtain mutants of Aspergillus niger 20 for raising citric acid

- 16 -

(CA) production. The uv treatment resulted in the development of

31 isolates, some of which differed in their morphology and

sporulation ability. Irradiation for 12 min. did not improve CA

yield by the developed mutants. Nine mutants gave significantly

higher yields of CA than parent culture when exposed to uv

treatment for 18 min. (6 mutants) and 24 min. (3 mutants). The

highest yield of CA obtained after exposure 18 min. (26.08%

higher than the parent culture) was that produced by isolate 5 uv

18. Irradiation for 24min. appeared to be the best treatment for

inducing mutation with isolate 19 uv 24 which produced a

32.13% higher CA yield than the parent control. When these nine

active mutants were tested for their CA productivity in the

presence of potassium ferrocyanide, only the isolates 5 uv 18 and

21 uv 24 each yielded 3% more CA than the original culture on a

sugar basis.

Rugsaseel et al., 1993 induced mutants with enhanced

citric acid production from soluble starch from Aspergillus niger

wu-2223L. after uv irradiation of a conidia suspension of strain

wu-2223L, mutants were selected on modified starch-methyl red

agar plates on the basis of higher amylolytic activity and acid

productivity. The 8 mutants selected showed enhanced citric acid

production from soluble starch in shaking culture. Among them, a

representative mutant strain, 2M-43, produced 48.0 g/L of citric

acid from 120 g/L of soluble starch in qd of cultivation in shaking

- 17 -

culture, whereas strain wu-2223L produced 35.1 g/L.

Glucoamylase activities in the culture filtrates of strains 2M-43

and wu-2223L reached maximum level of 3.62 u/ml and 2-11

u/ml, respectively, both at 3d of cultivation, and thereafter

decreased.

Sarangbin and Watanapokasin, 1999 carried out

selection of protease-negative mutant strains of A. niger in semi-

solid culture in order to enhance citric acid production from yam

bean. The protease-negative mutants were obtained by uv

irradiation of the parental strain Yang no. 2 using a halo selection

medium, a number of mutants with decreased extra cellular

protease activity were selected. Citric acid productivity by the

selected mutant strains was tested on a modified starch-methyl red

agar plate. The best mutant strain Yw-122 was obtained and

produced 106 g/L of citric acid, whereas the parental strain Yang

no 2 produced 58 g/L, from 140 g/L of soluble starch in semi-

solid culture at 5 days of cultivation time. During the whole

period of cultivation when 17 g/ plate of red-shaped yam bean

was used instead of soluble starch, the protease-negative mutant

strain Yw-112 produced 490 g/L of citric acid, which is

approximately 1.5 times as much citric acid as Yang no 2

produced.

- 18 -

Conte & Marine, 2003 induced mutants of Aspergillus

niger N402, by uv mutagenesis, were selected and tested for

resistance or sensitivity to 5-fluorocytosine. Some mutants

showed increased citric acid production, which did not correlate

with the intracellular amount of protein or ammonium ion. The

resistance to 5-fluorocytosine proved to be a rational approach for

isolation of new mutants with improved production of citric acid.

The best mutant (FR13) accumulated double the amount of citric

acid produced by the parental strain. Pewlong et al., 2003 used

ultraviolet and gamma irradiation to induce mutation of A. niger

ATCC11414 to increase ability of citric acid production. Five

mutants of high-producing citric acid were 7 uv-18, A2-14, 9 uv-2,

9 uv 27 and 9 uv-10. The yield of citric acid was 2.0 to 3.84 fold

higher than that of the wild type strain.

Goulart and Marin, 2005 studied some filamentous fungi

present the phenomenon of dimorphism, their morphological

structure alterations being capable of including metabolism

changes. The A. niger strain 10v10, a producer of citric acid, was

submitted to the mutagenic action of ultraviolet irradiation which

respectively selects mutants sensitive or resistant to the antifungal

agent 5-fluorcytosine (5-FC). 5-FC sensitive mutants presented a

morphological alteration to a yeast-like form. Morphological

reversal to the filamentous form was observed only. The presence

- 19 -

of Ca Cl2 (500 mµ) for the mutants strains/ and 2 while the acid

production occurred in both, yeast-like and filamentous forms.

Rodrigues et al., 2010 obtained eleven mutant strains with

uv irradiation of A. niger LPBBC, tested in SSF where two

mutants showed a higher citric acid (CA) production when

compared to the parental strain, A. niger LPBB produced 537.6 g

of citric acid / kg of citric pulp (CP) on the sixth day of

fermentation, while A. niger LPBB6 produced 616.5 g of CA/ kg

of CP on fourth day of fermentation representing a 19.5%. and

37% gain, CA production has been conducted through a careful

strain selection, physical-chemical optimiza-tion and mutation.


acid through γγγγ rays:

Das & Nandi, 1972 treated Aspergillus niger with uv and

gamma irradiation (60Co). Mutagenic treatments resulted in

substrains with either increased or decreased citric acid

production, while some were unchanged in this respect. Low-

yielding strains predominant in all the experiments. Analysis of

the superior substrains showed that gamma irradiation was most

effective in producing the greatest number of superior mutants.

Islam et al., 1984 investigated semi-pilot scale production

of citric acid with a-gamma-ray induced mutant (HB3) of

Aspergillus niger using 500, 1000 and 1500ml medium in 51

- 20 -

fermentation jars. Yield of citric acid was found to be seven-fold

higher compared to the parent in 1000ml medium and the

corresponding increase was two-fold in the 500ml medium. With

1500 ml/ formation jar the yield was low with both the parent and

the mutant strain, through the mutant gave higher yield compared

to the parent. Islam, 1990 subjected conidia of the citric acid

fermenting fungal mutant Aspergillus niger 14/20 to the gamma

radiation treatment. Mutants giving higher total triable acid values

than their parent strain were selected and tested under varied

environmental and cultural conditions. After screening of about

2000 mutants, two of them with the highest yield of citric acid in

molasses medium (60-76 gm/ml) were selected for further

experiments.

Begum et al., 1990 induced mutants of A. niger by γ-rays

in different carbohydrate media, they isolated a natural strain of

A. niger CA16, and two of its second step mutants, 136/40 and

277/30, grown on different sugar substrates gave maximum citric

acid yields of 34, 70, and 126 mg/ml respectively in sucrose

medium. Combination of two sugars in the medium at 50% of

each improved the yields of citric acid for the sucrose: glucose,

glucose: sorbitol, glucose: xylose and xylose: sorbitol

- 21 -

combinations with the mutant strains. Inclusion of galactose in

combinations decreased the citric acid yield.

Begum et al., 1991 studied effect of cultural pH and

incubation temperature on citric acid yield and kinetic patterns of

citric acid fermentation by a natural isolate of A. niger as CA 16

and one of its gamma ray induced mutants using cane molasses as

growth and fermentation substrate. Mutant strain, 277/30 gave

maximum citric acid yield of 35 g/L at pH 3.5 and 28oC in

molasses medium. Parent strain, CA 16 gave a maximum yield of

34 g/L at pH 4.0 and 26oC in molasses medium adjusted to 16%

sugar and 100% Prescott salt in the medium. In kinetic studies,

strains showed combinations kinetics of citric acid fermentation

where product formation is directly related to growth and cell

mass and indirectly related to carbohydrate uptake.

El-Batal et al., 1995 exposed inocula of A. niger

EMCC111 to doses (0.05 – 0.8 KGy) of γ-ray and showed that the

dose 0.4 kGy was the optimum for maximum citric acid

production. Parvez et al., 1998 studied citric acid production

from sugar cane molasses by A. niger NTAB 280 in a batch

cultivation process. A maximum of 90 g/L total sugar was utilized

in citric acid production medium. From the parental strain A.

niger, mutant strains, showing resistance to 2-deoxyglucos in

- 22 -

vogal's medium containing molasses as a carbon source were

induced by γ-irradiation. Among the new series of mutant strains,

strain RP7 produced 120 g/L while the parental strain produced

80 g/L citric acid (1.5 fold improvement) from 150 g/L of

molasses sugars. The period of citric acid production was

shortened from 10 d for the mild-type strain to 6-7 d for the

mutant strain. Also the mutant grew faster than its parent. This

indicated that the selected mutant is insensitive to catabolite

repression by higher concentrations of sugars for citric acid

production.

- 23 -

Materials and Methods

Microorganisms (parental strains):

Twenty strains of Aspergillus niger (wild or parental

strains) were isolated from different rotted sources.

Media:

I. Potato-dextrose agar medium (PDA) was used for isolation.

Strain of A. niger were grown on PDA medium, incubated

at 28°C for 7 days, this medium also used for purification

and maintenance the strains.

II. The indicator medium used for screening the strains and

citric acid production as described earlier (Chopra et al.,

1983). This medium contained (g/l) glucose 20.0, KH2

Po4,1.0; Mg So4, 7H2O, 0.25; NH4 No3, 2.50; pH, 5.5;

bromocresol purple (0.4% in alcohol), 10ml; agar agar,

20.0; the basal liquid medium used for production of citric

acid refer to the indicator medium minus carbon source,

agar-agar and the bromocresol purpule.

Inoculum preparation:

Spore suspension was prepared from 7 days old culture of

parental A. niger strains with sterilized water with shaking

vigorously for 1 min, the spore density was adjusted to

- 24 -

107spores/ml of the suspension (Mourya & Jauhri 2000 and

Ikram et al., 2004).

Carbohydrate by-products:

Maize straw and potato solid wastes were cut to small

pieces, sugar beet pulp was grounded to very small pieces (Baig

et al., 2004) and molasses diluted by water (1:3). These substrates

used without any treatment for growing all the strains alone at

different concentrations i.e. 5,10,15,20 & 25%, without any

additions, and also used for growing all the strains as carbon

source instead of glucose in basal liquid medium.

Cultivation:

A niger strains allowed to grow in 100ml fermentation

medium dispensed in 250ml Erlenmeyer flasks. 1 ml of 107 spores

were used to inoculate the flasks, then incubated at 30°C. Samples

were taken at different time intervals i.e. 6 and 12 days and tested

for citric acid production (Mourya & Jauhri 2000 and Ikram et

al., 2004).

Exposure of selected parental strains of Aspergillus niger to

uv light irradiation and γγγγ-rays:

A spore suspension was prepared (107 spores/ml), 0.1ml of

spore suspension was exposed to uv irradiation (wavelength 320

nm) at a distance of 30cm with exposure times of 2, 5, 10, 15, 20,

- 25 -

25 & 30 min. The uv lamp was used in dark environment in order

to avoid the repair mechanism that could invert the mutagenic

effects (Zaha 2003 and Rodrigues et al., 2010).

The spore suspension was also exposed to different does of

gamma rays i.e., 25, 50, 100, 200 Gy by Co-60 gamma indicator

(dose rate 11.1Gy/min) (Golubtsova et al., 1978, 1976&1972

and El-Batal et al., 1995) located at cyclotorn at Nuclear

Research Center, Inshas, Egypt.

Isolation of obtained strains (treated isolates):

Spore suspensions (0.1ml) were subjected to uv and gamma

rays, then plated on the indicator medium. The plates were

wrapped in carbon paper and incubated in dark at 30°C for a

period of 48 h. Colonies appeared in plates of each treatment were

obtained and tested for citric acid production (Mourya and

Jauhri 2000).

Assay of citric acid:

Citric acid was estimated gravimetrically, using pyridine-

acetic anhydride method as reported by (Marrier & Boulet, 1958

and Ikram et al., 2004). One ml of the culture filtrate along with

1.3ml of pyridine was added in the test tube and shacked then

5.70ml of acetic anhydride was added in the test tube. The test

tube was placed in a water bath for 30min. The optical density

- 26 -

was measured on a spectrophotometric (405 nm) and citric acid

contents of the sample was estimated with reference (run parallel,

replacing 1.0 ml of the culture filtrate with distilled water) to the

standard. Data recorded are the mean of three replicates ±

standard deviation.

0.0 0.2 0.4 0.6 0.8 1.0

0.1

0.2

0.3

0.4

0.5

0.6

Fig.(1): Standard curve citric acid.

Optical density

Conc. of citric acid, (mg/ml)

Random Amplified Polymorphic DNA Polymerase Chain

Reaction (RAPD-PCR):

There were a number of standard protocols by which

specific DNA sequences can be subjected to successive

amplification. One of the most reliable techniques currently used

in molecular biology is DNA amplification by PCR, which is a

- 27 -

procedure that allows rapid detection of the presence or absence

of a target DNA sequence in any genetic material. In this

technique, DNA is amplified in vitro by a series of polymerization

cycles consisting of three temperature-dependent steps

(Denaturation, annealing and extension) resulting in target DNA

amplification (Mullis et al., 1986 and Rychlik et al., 1990).

Nearly ten years ago, anew genetic assay was developed

indepently by two different laboratories (Welsh & Neclellend,

1990 and Williams et al., 1991). This procedure, which we have

called the RAPD assay, detects nucleotide sequence

polymorphisms in a DNA amplification-based as say using only a

single primer of arbitrary nucleotide sequence. In this reaction, a

single species of primer binds to the genomic DNA at two

different sites on opposite strands of the DNA template.

DNA was isolated using Maxweld 16 (promega) instrument

according to the manufacture instructions.

RAPD-PCR

Reactions were performed in a total volume 50µl reaction

buffer (100 mM KC1, 100 mm Tris HC1 pH 8.3) 3.0mm MgCl2,

200mm dNTPs (Promega Biotec. Inc.) 50p/mole primers and 0.2

µl Taq Polymerase (Hot Start). This reaction was added to 0.1µl

genomic DNA. All reactions tubes, pipette tips, micro pestles and

- 28 -

water were irradiated with uv light to destroy possible

contaminating surface DNA (Ou et al., 1991). Irradiation

treatment was 20 minutes at 2.5cm from the bulbs of a Gene

linker (Biorad, inc.) uv light source.

Tubes contain mixes were placed in a thermcycler (Perkin-

Elmer 2400) and DNA was amplified using the following

temperature cycle modified from (Black et al., 1992).

Preparation of PCR Reactions

The reactions were carried out in a volume of 50µl

containing 250ng of genomic DNA template, 2.5mM MgCl2,

1- A master mixture for 12 reactions (10 PCR wells) was

prepared in 1.5 ml microcentrifuge tube, so that each reaction

contained.

Component Amount for one PCR reaction

H2O 35.1µl

10 reaction buffer 5µl

DNTPs mix (100 mM) 1µl

2- An aliquot of 43.6 µl master mix was dispensed in each PCR

tube (o.5ml).

3- 5 µl of the primer (10 p/mole) were added to each tube.

4- 1 µl of the template (250 ng) was added.

5- 0.2 µl of Hot start Taq (1.25 Unit) was added.

- 29 -

PCR Program and temperature profile

Amplification of the DNA was performed by placing the

tubes containing the reactions in a Perkin Elmer thermal cycler

2400.

RAPD PCR performed in 50 ul reaction volumes for 30

cycles. After the reaction mixture was mixed with DNA loading

buffer and electrophoresed on 1% Agarose gel.

Temperature profile

Step 0: 94°C 5 min

Step l: 94°C 40 sec

Step 2: 36°C 1 min

Step 3: 72 °C 2 min

Step 4: 72 °C 7 min

Step 5: 4°C Hold

Protein electrophoresis

Sodium dodecyl sulfate (SDS) - Polyaciylamide gel

It was carried out at Agricultural Research Center,

Agricultural Genetic Engineering Research Institute.

Discontinuous Polyacrylamide gels consist of a resolving or

separating (lower) gel and stacking (upper) gel. The stacking gel

acts to large sample volumes, resulting in better band resolution

than, if possible, using the same volumes on a gel without a

stacJc. Gels were prepared according to (Laemmli, 1970).

- 30 -

Preparation of separating gel (10%)

Distilled water 4 ml

1.5 MTris-HCI 2.50 ml

10% SDS 100µl

Acrylamide/bis (30%) 3.33 µl

10% APS 50 µl

TEMED 5 µl

Volume required to completely fill gel sandwich may be adjusted

depending on application.

Preparation of stacking gel (4.0%)


0.5MTris-HCI 1.25ml

10%SDS 50 µl

Acrylamide/bis (30%) 670 µl

10% APS 25 µl

TEMED 5 µl

To prepare monomer solution of both separating and

stacking gels, combine all reagents, except the TEMED and

ammonium persulphate (APS), and degas under vacuum for 10

minutes. Add the two catalysis just prior to casting the gels. The

amount of stacking gel may be adjusted depending on the height

of the separating gel. Higher TEMBD concentration for faster

- 31 -

polymerization is required for the stacking gel because of the

inhibitory effect of atmospheric oxygen associated with the comb.

Stock solutions

Acrylamide/bis (30%)

Acrylamide 29.2 g

N-N-bis-methylene-acrylami 0.8 g

Make to 100 ml with distilled water. Filter and store at 4°C in

dark (30 days maximum).

1.5 M Tris-HCI

Trisbase 18.15g Distilled water 60 ml

Adjust pH 8.8 with IN HC1. Complete the volume to 100 ml with

distilled water and store at4°C.

0.5M Tris-HCI

Tris base 6g


Adjust to pH 6.8 with IN HC1, complete the volume to 100

ml with distilled water and store at 4°C.

10% SDS

Dissolve 10 g SDS in warm water with gentle stirring and

bring the volume to 100 ml with distilled water.

- 32 -

10 % ammonium persulphate (APS)

Dissolve 100 mg APS in 1 ml distilled water.

Ammonium sulfate {(NH4)2SO4=132.13 MWT} Provided by

BDH laboratory supplies Poole, BH 15 LTD, England

{Tel: (01202) 669700}.

Treatment buffer (62.5 mM Tris, 20% Glycerol, 2% SDS &

5% B-ME)

Distilled water 4.0 ml

0.5MTris-HCl 1.0ml

Glycerol 0.8 ml

10% SDS 1.6ml

B-ME 0.4 ml

0.05% (W/V) Bromophenol blue 0.2 ml

5% Tank buffer (IX = 25 mM Tris, 192mM glycine and 0,1%

SDS)

Tris base 15 g

Glycine 72 g

SDS 5 g

Complete to one liter with distilled water and store at 4°C.

The working solution is IX with pH 8.3. Staining solution

Coomassie blue R-250 0.1 %

Methanol 40 %

Glacial acetic acid 10%

- 33 -

Destaining solution

Glacial acetic 10%

Methanol 40%

Completed to 100m of distilled water

Protein banding patterns

Preparation and solubilization of the total proteins

Total proteins of the four isolates of Aspergillus niger were

analyzed by SDS-PAGE. Pellets were collected by centrifugation

at 12000 rpm at 4°C, washed once with distilled water and then

with 1 ml of ImM NaCl containing 5 mM EDTA. Proteins were

separated on the basis of molecular weight by Sodium Dodecyl

Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

(Laemmli,1970), then heated in the present of low molecular

weight thiol (2-mercaptoethanol) and SDS denatured total cellular

protein from vegetative or sporulated cells. One volume of the

cell suspension was mixed with one volume of 2X treatment

buffer (0.25M Tris-HCL PH6.8, 4% SDS, 20% glycerol and 10%

2-mercaptoethanol) and boiled in a water bath for 90 seconds then

quickly transferred to ice water and kept until loading the gel.

Protein electrophoresis

The gel apparatus was assembled and the lower and upper

chambers were filled with the tank buffer. A drop of

- 34 -

Bromophenol blue was added as a tracking dye. A Hamilton

syringe was used to load equal amounts of proteins (25 M1) in

each well. High range molecular weight protein marker from Bio-

Rad was used. Electrophoresis was carried out at about 100 volts

in Ix Tris/glycine- SDS-running buffer. After electrophoresis, the

gel was stained in 50ml of staining solution (0.125% Coomassie

blue R-250, 50% methanol and 10% acetic acid). The gel was

placed between two sheets of cellophane membrane and dried on

gel drier for 2hrs and photographed.

Preparation and casting of the SDS-PoIyacrylamide Gel

The gels were prepared from monomer solution of 30%

Acrylamide and 2.7% Bis-Acrylamide. The gels were prepared as

separating gel containing 10% acrylamide monomer and stacking

gel 4%. Ammonium persulphate and TEMED were used as

initiators for cross-linking and polymerization. The details of the

solution are mentioned in the material section. The components of

the separating gel solution were placed in 125ml side arm vacuum

flask, stoppered flask and apply vacuum for several minutes to

insure suction of oxygen from the solution. Ammonium

persulphate and TEMED were added and the flask gently swirled

to mix the solutions being careful not to generate babbles. The

solution was pipetted into the assembled vertical slab gel unit in

the casting mode to level 1.5cm from the top. N-butanol was

- 35 -

layered on the top of the solution. The gel was left to polymerize

at room temperature for two hours. Prior to addition of stacking

gel, the N-butanol was poured from the surface of the gel and the

surface was washed once with overlay buffer. The 4% stacking

gel was prepared by mixing its components as indicated in

materials section, degassing under vacuum, adding TEMED and

ammonium persulphate and gently swirling the mixture. Stacking

solution was added to the top of the separating gel, combs were

inserted and the gel was allowed to polymerize for at least half an

hour. After polymerization the combs were removed slowly from

the gel and each well was rinsed with tank buffer using a

Hamilton syringe.

- 36 -

- 37 -

Results

Twenty strains of Aspergillus niger were isolated from

different sources (Table 1) they were screened for their capacity

to produce citric acid on indicator medium and different

carbohydrate by-products:


indicator medium:

Data recorded in Table (2) showed that all the strains under

the study were able to produce citric acid on indicator medium as

indicated by the diameter of the clear zone (mm) and production

in different quantities at different time intervals i.e. 4, 8 and 12

days. Data showed that the best incubation period for production

was 12 days for all isolates. Maximum production on indicator

medium after 12 days were 0.95, 0.94 & 0.99mg/ml for A1, A4 &

A5 respectively (Figs. 2& 3) while the lowest production recorded

in this medium were 0.74, 0.71, 0.68 & 0.62 mg/ml for A8, A16,

A18 & A19 respectively (Figs. 2 & 4).

- 38 -

Table (1): Source of isolation of twenty strains of A. niger.

Strains of A. niger Source of rotted material

A1 Maize grains

A2 Chesse

A3 Peanut

A4 Banana

A5 Apple

A6 Potato

A7 Bread

A8 Phylloplane of Solanum persicum

A9 Orange

A10 Peach

A11 Bagasse

A12 Tomato

A13 Peach

A14 Pickles

A15 Yoghurt

A16 Garlic

A17 Lemon

A18 Potato

A19 Tomato sauce

A20 Bread

- 39 -

Table (2): Diameter of clear zone (mm) and production of

citric acid (mg/ml) by different strains of Aspergillus

niger using indicator medium at different time

intervals.

Diameter of clear zone (mm) Citric acid production (mg/ml)

Isolates

4 days 8 days 4 days 8 days 12 days

A1 50 90 0.38±±±±0.04 0.67±±±±0.03 0.95±±±±0.08

A2 35 55 0.25±±±±0.09 0.56±±±±0.06 0.81±±±±0.09

A3 45 80 0.36±±±±0.07 0.65±±±±0.10 0.91±±±±0.13

A4 48 85 0.38±±±±0.03 0.66±±±±0.05 0.94±±±±0.07

A5 51 92 0.39±±±±0.06 0.68±±±±0.08 0.99±±±±0.12

A6 25 45 0.21±±±±0.07 0.52±±±±0.04 0.76±±±±0.10

A7 27 47 0.24±±±±0.09 0.53±±±±0.09 0.77±±±±0.06

A8 30 38 0.20±±±±0.09 0.50±±±±0.09 0.74±±±±0.04

A9 37 70 0.31±±±±0.12 0.60±±±±0.11 0.88±±±±0.11

A10 40 75 0.34±±±±0.04 0.62±±±±0.07 0.88±±±±0.08

A11 36 55 0.26±±±±0.05 0.56±±±±0.06 0.82±±±±0.06

A12 35 60 0.27±±±±0.03 0.57±±±±0.06 0.84±±±±0.07

A13 23 42 0.20±±±±0.09 0.51±±±±0.05 0.76±±±±0.09

A14 38 65 0.29±±±±0.10 0.59±±±±0.13 0.85±±±±0.09

A15 37 65 0.29±±±±0.11 0.59±±±±0.06 0.86±±±±0.11

A16 22 30 0.20±±±±0.09 0.48±±±±0.08 0.71±±±±0.04

A17 39 72 0.32±±±±0.09 0.61±±±±0.04 0.90±±±±0.07

A18 25 30 0.19±±±±0.07 0.47±±±±0.09 0.68±±±±0.03

A19 17 22 0.17±±±±0.03 0.45±±±±0.05 0.62±±±±0.05

A20 38 62 0.26±±±±0.03 0.58±±±±0.07 0.85±±±±0.08

- 40 -

- 41 -

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

citric acid production (mg/ml)

A1 A4 A5

A. niger isolates

4 days

8 days

12days

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


A 8 A 16 A 18 A 19

Weak isolates

4 days

8 days

12days

Fig. (3): Maximum Citric acid production by A. niger isolates (A1, A4 &

A5) on indicator medium at different time intervals.

Fig. (4): Weakest production of citric acid by four isolates (A8, A16, A18

& A19) on indicator medium at different time intervals.

- 42 -


maize straw after 12 days incubation:

Table (3) showed citric acid production (mg/ml) by A.

niger strains when allowed to grow on different concentrations of maize straw i.e., 5, 10, 15, 20 & 25% without any additions after 12 days incubation. Maximum production were 0.68, 0.64 & 0.70 mg/ml by A1, A4 & A5 respectively at 25% concentration. It can be noticed from the table that increasing the concentration of maize straw led to increase citric acid production by the isolates. The best concentration for production was 25%. The lowest production at 25% maize straw obtained by A8, A16, A18 & A19, the production were 0.48, 0.47, 0.47 & 0.46 mg/ml respectively (Table 3 & Fig. 5).

Data in table (4) showed citric acid production (mg/ml) by Aspergillus niger isolates when allowing to grow on basal medium amended with the previous mentioned concentrations of maize straw after 12 days incubation. Maximum production recorded were 0.73, 0.70 & 0.84mg/ml by A1, A4 & A5 respectively at 25% concentration (Fig. 6). It can be noticed that the production of these isolates growing on a basal medium amended with maize straw was more than that produced by isolates grown on maize straw only. Also increasing substrate concentration led to increase citric acid production for all Aspergillus niger isolates. Weak isolates (A8, A16, A18 & A19) which grown on basal medium amended with maize straw showed rarely similar production levels to that of straw alone.

- 43 -

Table (3): Citric acid production (mg/ml) by different isolates

of A. niger grown on different concentrations of

maize straw after 12 days incubation.

Citric acid production (mg/ml)

Maize straw (%) Strain No.

5 10 15 20 25

A1 0.26±±±±0.07 0.31±±±±0.02 0.45±±±±0.07 0.55±±±±0.05 0.68±±±±0.04

A2 0.19±±±±0.02 0.25±±±±0.05 0.38±±±±0.05 0.40±±±±0.03 0.51±±±±0.07

A3 0.24±±±±0.06 0.28±±±±0.03 0.40±±±±0.06 0.53±±±±0.05 0.63±±±±0.03

A4 0.22±±±±0.08 0.30±±±±0.09 0.44±±±±0.03 0.54±±±±0.11 0.64±±±±0.05

A5 0.27±±±±0.03 0.33±±±±0.08 0.46±±±±0.07 0.58±±±±0.09 0.70±±±±0.09

A6 0.22±±±±0.05 0.24±±±±0.06 0.33±±±±0.09 0.39±±±±0.07 0.51±±±±0.08

A7 0.21±±±±0. 10 0.27±±±±0.04 0.34±±±±0.10 0.41±±±±0.10 0.53±±±±0.08

A8 0.16±±±±0.09 0.21±±±±0.07 0.31±±±±0.12 0.35±±±±0.09 0.48±±±±0.06

A9 0.20±±±±0.07 0.24±±±±0.03 0.34±±±±0.07 0.43±±±±0.05 0.59±±±±0.11

A10 0.20±±±±0.07 0.25±±±±0.09 0.37±±±±0.05 0.50±±±±0.06 0.62±±±±0.12

A11 0.20±±±±0.06 0.24±±±±0.12 0.37±±±±0.09 0.40±±±±0.11 0.53±±±±0.10

A12 0.20±±±±0.03 0.24±±±±0.04 0.31±±±±0.08 0.41±±±±0.05 0.55±±±±0.04

A13 0.17±±±±0.03 0.22±±±±0.05 0.32±±±±0.11 0.38±±±±0.07 0.50±±±±0.05

A14 0.23±±±±0.12 0.24±±±±0.11 0.33±±±±0.09 0.44±±±±0.04 0.56±±±±0.07

A15 0.23±±±±0.07 0.23±±±±0.08 0.32±±±±0.09 0.44±±±±0.08 0.56±±±±0.08

A16 0.15±±±±0.09 0.20±±±±0.04 0.29±±±±0.08 0.32±±±±0.09 0.47±±±±0.04

A17 0.21±±±±0.06 0.23±±±±0.07 0.35±±±±0.08 0.47±±±±0.05 0.61±±±±0.03

A18 0.15±±±±0.09 0.21±±±±0.05 0.30±±±±0.04 0.33±±±±0.07 0.47±±±±0.03

A19 0.13±±±±0.11 0.17±±±±0.04 0.27±±±±0.07 0.30±±±±0.07 0.45±±±±0.05

A20 0.20±±±±0.03 0.22±±±±0.03 0.30±±±±0.09 0.44±±±±0.03 0.56±±±±0.07

- 44 -


of A. niger growing on basal medium amended

with different concentrations of maize straw after

12 days incubation.


Maize straw (%) Strain No.

5 10 15 20 25

A1 0.35±±±±0.07 0.48±±±±0.05 0.61±±±±0.3 0.70±±±±0.08 0.73±±±±0.04

A2 0.28±±±±0.04 0.39±±±±0.08 0.45±±±±0.07 0.51±±±±0.06 0.56±±±±0.03

A3 0.31±±±±0.07 0.51±±±±0.09 0.57±±±±0.05 0.60±±±±0.03 0.69±±±±0.07

A4 0.35±±±±0.06 0.45±±±±0.12 0.59±±±±0.04 0.65±±±±0.03 0.70±±±±0.09

A5 0.36±±±±0.06 0.53±±±±0.05 0.62±±±±0.04 0.73±±±±0.07 0.84±±±±0.11

A6 0.24±±±±0.09 0.33±±±±0.07 0.42±±±±0.03 0.50±±±±0.09 0.53±±±±0.08

A7 0.23±±±±0.12 0.31±±±±0.06 0.42±±±±0.08 0.49±±±±0.09 0.55±±±±0.05

A8 0.21±±±±0.08 0.30±±±±0.09 0.38±±±±0.06 0.41±±±±0.03 0.48±±±±0.03

A9 0.29±±±±0.08 0.39±±±±0.09 0.48±±±±0.07 0.53±±±±0.08 0.61±±±±0.07

A10 0.31±±±±0.04 0.41±±±±0.08 0.53±±±±0.09 0.57±±±±0.09 0.67±±±±0.06

A11 0.28±±±±0.03 0.38±±±±0.04 0.47±±±±0.06 0.54±±±±0.04 0.57±±±±0.09

A12 0.27±±±±0.07 0.34±±±±0.11 0.44±±±±0.05 0.51±±±±0.04 0.58±±±±0.08

A13 0.21±±±±0.09 0.30±±±±0.06 0.37±±±±0.05 0.42±±±±0.08 0.51±±±±0.03

A14 0.20±±±±0.03 0.32±±±±0.06 0.45±±±±0.04 0.53±±±±0.07 0.59±±±±0.09

A15 0.21±±±±0.03 0.33±±±±0.09 0.45±±±±0.11 0.53±±±±0.06 0.59±±±±0.04

A16 0.17±±±±0.05 0.22±±±±0.04 0.33±±±±0.03 0.40±±±±0.06 0.48±±±±0.11

A17 0.35±±±±0.08 0.40±±±±0.03 0.54±±±±0.03 0.53±±±±0.03 0.65±±±±0.010

A18 0.18±±±±0.06 0.23±±±±0.05 0.35±±±±0.13 0.40±±±±0.07 0.48±±±±0.04

A19 0.14±±±±0.09 0.19±±±±0.05 0.30±±±±0.05 0.33±±±±0.09 0.46±±±±0.07

A20 0.28±±±±0.11 0.35±±±±0.07 0.44±±±±0.12 0.51±±±±0.11 0.58±±±±0.03

- 45 -

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger growing on 25% maize straw

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger growing on basal medium amended with 25% of maize straw

Fig. (5): Citric acid production (mg/ml) by different isolates of A. niger

growing on 25% maize straw after 12 days incubation.


growing on basal medium amended with 25% maize straw

after 12 days incubation.

- 46 -


potato solid wastes (peels) after 12 days incubation:

Increasing concentration of potato solid wastes led to

increase citric acid production for all Aspergillus niger strains

(tables 5 & 6). The best concentration for production was 25%,

maximum production were obtained by A1 & A4, A5 after 12

days incubation. Production of these isolates on 25% potato solid

wastes were 0.75, 0.74 & 0.76mg/ml respectively (table 5 & Fig.

7) while the yield on basal medium amended with 25% potato

solid wastes were 0.94, 0.91 & 0.98mg/ml respectively (table 6 &

Fig. 8). The production for all the strains on the basal medium

amended with potato solid wastes showed an increase over that

with potato solid wastes only. Lowest production recorded with

A8, A16, A18 & A19 in case of growing on both potato solid wastes

only and on basal medium amended with that carbohydrate by

product as shown from the two tables (5&6).


sugar beet pulp after 12 days incubation:

The highest production obtained by A1, A4 & A5, when

allowing to grow on sugar beet pulp & also on basal medium

amended with that carbohydrate by- product (tables 8 & 9). The

production on 25% sugar beet pulp were: 1.10, 1.05 & 1.08

mg/ml respectively (table 8 & Fig. 9). While the production on

basal medium amended with sugar beet pulp were: 1.56, 1.46 &

1.52 mg/ml respectively (Fig. 10).

- 47 -


of A. niger grown on different concentrations of

potato solid wastes after 12 days incubation.


Potato solid wastes (%) Strain No.

5 10 15 20 25

A1 0.37±±±±0.11 0.48±±±±0.08 0.55±±±±0.04 0.67±±±±0.07 0.75±±±±0.06

A2 0.23±±±±0.04 0.35±±±±0.07 0.44±±±±0.12 0.53±±±±0.10 0.60±±±±0.08

A3 0.33±±±±0.09 0.46±±±±0.10 0.54±±±±0.02 0.64±±±±0.04 0.71±±±±0.05

A4 0.35±±±±0.03 0.47±±±±0.07 0.54±±±±0.08 0.65±±±±0.04 0.74±±±±0.09

A5 0.38±±±±0.07 0.49±±±±0.09 0.57±±±±0.03 0.68±±±±0.04 0.76±±±±0.07

A6 0.22±±±±0.11 0.31±±±±0.08 0.48±±±±0.07 0.57±±±±0.06 0.59±±±±0.04

A7 0.22±±±±0.07 0.32±±±±0.05 0.47±±±±0.03 0.56±±±±0.09 0.59±±±±0.05

A8 0.21±±±±0.08 0.28±±±±0.09 0.42±±±±0.04 0.51±±±±0.03 0.56±±±±0.03

A9 0.28±±±±0.06 0.44±±±±0.07 0.51±±±±0.08 0.57±±±±0.04 0.65±±±±0.07

A10 0.30±±±±0.06 0.45±±±±0.05 0.53±±±±0.03 0.61±±±±0.04 0.68±±±±0.03

A11 0.23±±±±0.09 0.29±±±±0.03 0.44±±±±0.06 0.57±±±±0.03 0.61±±±±0.09

A12 0.24±±±±0.04 0.40±±±±0.03 0.47±±±±0.07 0.59±±±±0.05 0.63±±±±0.10

A13 0.21±±±±0.07 0.30±±±±0.06 0.45±±±±0.09 0.51±±±±0.12 0.57±±±±0.9

A14 0.27±±±±0.03 0.42±±±±0.07 0.49±±±±0.09 0.58±±±±0.14 0.64±±±±0.11

A15 0.26±±±±0.03 0.42±±±±0.09 0.47±±±±0.12 0.57±±±±0.08 0.64±±±±0.04

A16 0.20±±±±0.11 0.25±±±±0.07 0.43±±±±0.03 0.50±±±±0.09 0.55±±±±0.07

A17 0.29±±±±0.09 0.45±±±±0.06 0.52±±±±0.08 0.60±±±±0.05 0.67±±±±0.03

A18 0.20±±±±0.03 0.26±±±±0.09 0.43±±±±0.06 0.50±±±±0.07 0.53±±±±0.06

A19 0.18±±±±0.04 0.22±±±±0.08 0.40±±±±0.09 0.48±±±±0.03 0.51±±±±0.05

A20 0.25±±±±0.05 0.42±±±±0.70 0.49±±±±0.04 0.57±±±±0.03 0.64±±±±0.07

- 48 -

Table (6): Citric acid production (mg/ml) by different isolates of A. niger

grown on basal medium amended with different

concentrations of potato solid wastes after 12 days

incubation.


Potato solid wastes (%) Strain No.

5 10 15 20 25

A1 0.36±±±±0.05 0.58±±±±0.04 0.77±±±±0.07 0.85±±±±0.09 0.94±±±±0.03

A2 0.29±±±±0.07 0.48±±±±0.08 0.51±±±±0.06 0.55±±±±0.11 0.69±±±±0.09

A3 0.34±±±±0.03 0.56±±±±0.08 0.74±±±±0.04 0.78±±±±0.12 0.88±±±±0.10

A4 0.34±±±±0.08 0.57±±±±0.05 0.76±±±±0.07 0.80±±±±0.06 0.91±±±±0.08

A5 0.37±±±±0.06 0.58±±±±0.04 0.77±±±±0.08 0.89±±±±0.10 0.98±±±±0.09

A6 0.26±±±±0.03 0.37±±±±0.09 0.46±±±±0.08 0.54±±±±0.03 0.65±±±±0.07

A7 0.26±±±±0.07 0.38±±±±0.08 0.48±±±±0.04 0.56±±±±0.04 0.66±±±±0.03

A8 0.25±±±±0.10 0.36±±±±0.12 0.43±±±±0.03 0.54±±±±0.07 0.62±±±±0.08

A9 0.30±±±±0.08 0.41±±±±0.04 0.65±±±±0.03 0.69±±±±0.06 0.79±±±±0.05

A10 0.32±±±±0.07 0.43±±±±0.05 0.69±±±±0.06 0.75±±±±0.06 0.84±±±±0.12

A11 0.29±±±±0.03 0.47±±±±0.09 0.51±±±±0.09 0.55±±±±0.09 0.68±±±±0.09

A12 0.30±±±±0.07 0.46±±±±0.11 0.58±±±±0.04 0.63±±±±0.03 0.75±±±±0.04

A13 0.28±±±±0.05 0.37±±±±0.04 0.44±±±±0.03 0.54±±±±0.09 0.63±±±±0.03

A14 0.33±±±±0.04 0.48±±±±0.05 0.61±±±±0.07 0.65±±±±0.04 0.77±±±±0.05

A15 0.33±±±±0.03 0.49±±±±0.06 0.62±±±±0.09 0.66±±±±0.07 0.78±±±±0.05

A16 0.24±±±±0.03 0.37±±±±0.08 0.41±±±±0.09 0.52±±±±0.07 0.60±±±±0.07

A17 0.31±±±±0.08 0.51±±±±0.09 0.66±±±±0.10 0.70±±±±0.10 0.81±±±±0.11

A18 0.25±±±±0.09 0.38±±±±0.03 0.41±±±±0.013 0.51±±±±0.08 0.60±±±±0.09

A19 0.23±±±±0.09 0.35±±±±0.03 0.38±±±±0.04 0.49±±±±0.07 0.58±±±±0.05

A20 0.30±±±±0.11 0.43±±±±0.07 0.65±±±±0.05 0.71±±±±0.07 0.80±±±±0.06

- 49 -

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger grow ing on 25% of potato solid wastes

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger grow ing on basal medium amended w ith 25% of potato

solid wastes

Fig. (7): Citric acid production (mg/ml) by different isolates of A. niger growing

on 25% of potato solid wastes after 12 days incubation.

Fig. (8): Citric acid production (mg/ml) by different isolates of A. niger growing

on basal medium amended with 25% of potato solid wastes after 12

days incubation.

- 50 -

Increasing substrate concentrations led to increase the production

of the isolates, the best concentration for production under the study

was also at 25% in this carbohydrate by-product. Lowest production at

25% concentration on both sugar beet pulp and basal medium amended

with sugar beet pulp obtained by A8, A16, A18, A19. The productions of

these isolates on sugar beet pulp were 0.91, 0.72. 0.69, & 0.65mg/ml

while on basal medium amended with sugar beet pulp were: 0.94, 0.90,

0.85 & 0.82mg/ml respectively (table 8 & Fig. 10). It can be noticed that

citric acid production of the isolates on the basal medium amended with

sugar beet pulp was more than that if the isolates grown on sugar beet

pulp alone.


molasses after 12 days incubation:

Increasing molasses concentration under the study led to increase

the production of citric acid for all the isolates when allowing to grow on

both molasses only and on basal medium amended with molasses, the

best concentration for production was 25% (Tables 9 & 10). Highest

citric acid production recorded with: A1, A4 and A5 isolates in case of

growing on molasses only and also on basal medium amended with

molasses. Production of these isolates on 25% molasses only were 0.94,

0.84 and 0.86 mg/ml respectively (table 9). While on basal medium

amended with 25% molasses the production was 1.17, 1.04 & 1.15 mg/ml

respectively (table 10) while the lowest citric acid production obtained

by isolates A8, A16, A18 & A19, the production on 25% molasses were:

0.72, 0.67, 0.65 & 0.59 mg/ml respectively (Fig. 11), while the production

on basal medium amended with 25% molasses were: 0.80, 0.78, 0.76 &

0.65 mg/ml respectively (Fig. 12).

- 51 -


of A. niger growing on different concentrations of

sugar beet pulp after 12 days incubation.


Sugar beet pulp (%) Strain No.

5 10 15 20 25

A1 0.50±±±±0.12 0.68±±±±0.04 0.84±±±±0.05 0.91±±±±0.07 1.10±±±±0.09

A2 0.37±±±±0.06 0.52±±±±0.09 0.66±±±±0.04 0.79±±±±0.03 0.90±±±±0.10

A3 0.39±±±±0.07 0.63±±±±0.07 0.84±±±±0.08 0.91±±±±0.11 1.00±±±±0.04

A4 0.48±±±±0.09 0.69±±±±0.03 0.86±±±±0.06 0.93±±±±0.05 1.05±±±±0.03

A5 0.49±±±±0.04 0.70±±±±0.09 0.89±±±±0.09 0.92±±±±0.03 1.08±±±±0.08

A6 0.35±±±±0.03 0.60±±±±0.07 0.72±±±±0.12 0.83±±±±0.04 0.95±±±±0.11

A7 0.38±±±±0.13 0.65±±±±0.06 0.77±±±±0.10 0.88±±±±0.03 0.98±±±±0.09

A8 0.34±±±±0.05 0.48±±±±0.08 0.64±±±±0.03 0.74±±±±0.04 0.91±±±±0.07

A9 0.41±±±±0.06 0.53±±±±0.07 0.70±±±±0.07 0.83±±±±0.03 1.00±±±±0.09

A10 0.44±±±±0.09 0.58±±±±0.03 0.76±±±±0.07 0.80±±±±0.04 1.03±±±±0.08

A11 0.39±±±±0.06 0.57±±±±0.07 0.64±±±±0.10 0.82±±±±0.03 0.95±±±±0.03

A12 0.41±±±±0.09 0.59±±±±0.11 0.70±±±±0.18 0.85±±±±0.07 0.99±±±±0.04

A13 0.32±±±±0.03 0.45±±±±0.04 0.60±±±±0.09 0.73±±±±0.08 0.84±±±±0.07

A14 0.42±±±±0.04 0.63±±±±0.07 0.74±±±±0.03 0.89±±±±0.04 1.02±±±±0.09

A15 0.40±±±±0.08 0.60±±±±0.03 0.72±±±±0.03 0.84±±±±0.09 1.00±±±±0.06

A16 0.35±±±±0.11 0.44±±±±0.12 0.56±±±±0.09 0.65±±±±0.04 0.72±±±±0.07

A17 0.41±±±±0.07 0.60±±±±0.03 0.74±±±±0.10 0.87±±±±0.03 0.94±±±±0.04

A18 0.30±±±±0.04 0.40±±±±0.06 0.52±±±±0.05 0.64±±±±0.09 0.69±±±±0.10

A19 0.31±±±±0.03 0.38±±±±0.06 0.53±±±±0.07 0.60±±±±0.06 0.65±±±±0.09

A20 0.42±±±±0.05 0.59±±±±0.09 0.74±±±±0.07 0.89±±±±0.07 0.95±±±±0.11

- 52 -


of A. niger growing on basal liquid medium

amended with different concentrations of sugar

beet pulp after 12 days incubation.


Sugar beet pulp (%) Strain No.

5 10 15 20 25

A1 0.55±±±±0.06 0.75±±±±0.09 0.97±±±±0.07 1.15±±±±0.05 1.56±±±±0.03

A2 0.43±±±±0.08 0.69±±±±0.03 0.88±±±±0.04 0.93±±±±0.07 1.28±±±±0.03

A3 0.45±±±±0.09 0.75±±±±0.03 0.91±±±±0.09 0.97±±±±0.12 1.40±±±±0.04

A4 0.51±±±±0.05 0.72±±±±0.03 0.92±±±±0.04 1.06±±±±0.03 1.46±±±±0.05

A5 0.53±±±±0.11 0.73±±±±0.05 0.93±±±±0.09 1.09±±±±0.07 1.52±±±±0.06

A6 0.40±±±±0.09 0.66±±±±0.09 0.78±±±±0.03 0.89±±±±0.04 1.02±±±±0.09

A7 0.42±±±±0.09 0.71±±±±0.07 0.80±±±±0.07 0.90±±±±0.06 1.08±±±±0.06

A8 0.38±±±±0.03 0.57±±±±0.04 0.72±±±±0.06 0.80±±±±0.09 0.94±±±±0.07

A9 0.47±±±±0.09 0.69±±±±0.07 0.81±±±±0.03 0.92±±±±0.04 1.24±±±±0.08

A10 0.50±±±±0.04 0.73±±±±0.03 0.87±±±±0.06 0.96±±±±0.07 1.33±±±±0.09

A11 0.45±±±±0.08 0.62±±±±0.07 0.74±±±±0.03 0.90±±±±0.06 1.11±±±±0.04

A12 0.49±±±±0.11 0.64±±±±0.08 0.77±±±±0.09 0.92±±±±0.04 1.20±±±±0.07

A13 0.37±±±±0.12 0.53±±±±0.05 0.66±±±±0.014 0.84±±±±0.06 0.92±±±±0.05

A14 0.50±±±±0.06 0.71±±±±0.03 0.83±±±±0.04 0.94±±±±0.09 1.32±±±±0.02

A15 0.46±±±±0.05 0.65±±±±0.06 0.79±±±±0.07 0.91±±±±0.11 1.15±±±±0.04

A16 0.42±±±±0.07 0.51±±±±0.03 0.67±±±±0.04 0.86±±±±0.08 0.90±±±±0.06

A17 0.47±±±±0.06 0.69±±±±0.06 0.79±±±±0.07 0.92±±±±0.08 1.16±±±±0.09

A18 0.38±±±±0.12 0.54±±±±0.11 0.60±±±±0.08 0.70±±±±0.09 0.85±±±±0.04

A19 0.34±±±±0.09 0.46±±±±0.05 0.57±±±±0.07 0.67±±±±0.04 0.82±±±±0.03

A20 0.48±±±±0.04 0.67±±±±0.03 0.82±±±±0.09 0.93±±±±0.07 1.25±±±±0.08

- 53 -

0

0.2

0.4

0.6

0.8

1

1.2


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger grow ing on 25% of sugar beet pulp

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger grow ing on basal liquid medium amended w ith 25% of sugar

beet pulp


growing on 25% of sugar beet pulp after 12 days incubation.


growing on basal liquid medium amended with 25% of sugar

beet pulp after 12 days incubation.

- 54 -


of A. niger growing on different concentrations of

molasses after 12 days incubation.


Molasses (%) Strain No.

5 10 15 20 25

A1 0.45±±±±0.06 0.60±±±±0.08 0.75±±±±0.07 0.83±±±±0.04 0.94±±±±0.11

A2 0.31±±±±0.05 0.45±±±±0.03 0.52±±±±0.07 0.67±±±±0.08 0.77±±±±0.09

A3 0.35±±±±0.04 0.52±±±±0.09 0.70±±±±0.08 0.74±±±±0.04 0.82±±±±0.03

A4 0.30±±±±0.03 0.49±±±±0.04 0.70±±±±0.05 0.75±±±±0.07 0.84±±±±0.06

A5 0.43±±±±0.08 0.56±±±±0.07 0.71±±±±0.03 0.78±±±±0.03 0.86±±±±0.09

A6 0.27±±±±0.03 0.36±±±±0.05 0.49±±±±0.04 0.57±±±±0.05 0.76±±±±0.10

A7 0.29±±±±0.05 0.41±±±±0.08 0.52±±±±0.05 0.63±±±±0.08 0.77±±±±0.08

A8 0.30±±±±0.06 0.39±±±±0.07 0.59±±±±0.08 0.66±±±±0.06 0.72±±±±0.04

A9 0.38±±±±0.04 0.46±±±±0.14 0.62±±±±0.16 0.71±±±±0.03 0.83±±±±0.03

A10 0.42±±±±0.08 0.49±±±±0.09 0.67±±±±0.03 0.78±±±±0.05 0.87±±±±0.05

A11 0.34±±±±0.06 0.46±±±±0.10 0.53±±±±0.04 0.67±±±±0.03 0.87±±±±0.08

A12 0.39±±±±0.07 0.54±±±±0.07 0.66±±±±0.09 0.77±±±±0.04 0.89±±±±0.07

A13 0.26±±±±0.10 0.37±±±±0.07 0.51±±±±0.05 0.66±±±±0.03 0.72±±±±0.09

A14 0.35±±±±0.12 0.54±±±±0.03 0.65±±±±0.09 0.81±±±±0.07 0.98±±±±0.04

A15 0.33±±±±0.06 0.55±±±±0.07 0.61±±±±0.08 0.79±±±±0.03 0.87±±±±0.10

A16 0.28±±±±0.03 0.35±±±±0.09 0.49±±±±0.06 0.58±±±±0.09 0.67±±±±0.03

A17 0.39±±±±0.09 0.54±±±±0.04 0.69±±±±0.12 0.85±±±±0.10 0.90±±±±0.04

A18 0.24±±±±0.04 0.33±±±±0.07 0.44±±±±0.08 0.56±±±±0.09 0.65±±±±0.65

A19 0.23±±±±0.03 0.31±±±±0.08 0.45±±±±0.07 0.51±±±±0.06 0.59±±±±0.04

A20 0.38±±±±0.09 0.51±±±±0.03 0.64±±±±0.04 0.85±±±±0.10 0.90±±±±0.07

- 55 -

Table (10): Citric acid production (mg/ml) by different


medium amended with different concentrations of



Molasses (%) Strain No.

5 10 15 20 25

A1 0.52±±±±0.04 0.69±±±±0.03 0.87±±±±0.06 0.98±±±±0.09 1.17±±±±0.04

A2 0.39±±±±0.03 0.54±±±±0.05 0.63±±±±0.09 0.76±±±±0.07 0.92±±±±0.08

A3 0.43±±±±0.03 0.67±±±±0.09 0.82±±±±0.04 0.90±±±±0.07 0.98±±±±0.06

A4 0.41±±±±0.07 0.65±±±±0.09 0.84±±±±0.06 0.94±±±±0.09 1.04±±±±0.03

A5 0.51±±±±0.10 0.70±±±±0.07 0.87±±±±0.03 0.86±±±±0.04 1.15±±±±0.09

A6 0.30±±±±0.04 0.48±±±±0.03 0.61±±±±0.09 0.77±±±±0.08 0.85±±±±0.07

A7 0.34±±±±0.03 0.50±±±±0.11 0.66±±±±0.08 0.79±±±±0.03 0.87±±±±0.08

A8 0.33±±±±0.07 0.45±±±±0.07 0.63±±±±0.06 0.74±±±±0.12 0.80±±±±0.03

A9 0.45±±±±0.06 0.64±±±±0.05 0.74±±±±0.07 0.90±±±±0.05 1.02±±±±0.11

A10 0.50±±±±0.09 0.68±±±±0.10 0.79±±±±0.03 0.86±±±±0.03 1.09±±±±0.09

A11 0.40±±±±0.11 0.52±±±±0.09 0.64±±±±0.03 0.75±±±±0.13 0.94±±±±0.10

A12 0.43±±±±0.08 0.60±±±±0.08 0.71±±±±0.05 0.87±±±±0.03 0.99±±±±0.05

A13 0.31±±±±0.04 0.42±±±±0.05 0.60±±±±0.03 0.70±±±±0.06 0.78±±±±0.14

A14 0.41±±±±0.05 0.62±±±±0.03 0.80±±±±0.06 0.89±±±±0.16 1.03±±±±0.04

A15 0.40±±±±0.08 0.61±±±±0.13 0.76±±±±0.08 0.88±±±±0.04 0.97±±±±0.05

A16 0.31±±±±0.03 0.44±±±±0.09 0.59±±±±0.08 0.70±±±±0.08 0.78±±±±0.09

A17 0.48±±±±0.03 0.67±±±±0.08 0.78±±±±0.04 0.92±±±±0.09 1.05±±±±0.11

A18 0.28±±±±0.04 0.41±±±±0.07 0.56±±±±0.03 0.65±±±±0.06 0.76±±±±0.07

A19 0.29±±±±0.06 0.38±±±±0.04 0.50±±±±0.05 0.58±±±±0.10 0.65±±±±0.10

A20 0.44±±±±0.05 0.63±±±±0.08 0.78±±±±0.10 0.91±±±±0.05 1.00±±±±0.09

- 56 -

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger grow ing on 25% of molasses

0

0.2

0.4

0.6

0.8

1

1.2


A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

Isolates of A. niger grow ing on basal liquid medium amended w ith

25% of molasses


growing on 25% of molasses after 12 days incubation.


growing on basal liquid medium amended with 25% of


- 57 -

In conclusion from the above results (part I) it can be

noticed that type and concentration of carbohydrate by-products

under the study affect citric acid yield by Aspergillus niger

strains. Increasing carbohydrate by-product concentra-tion led to

increase the production of citric acid, the best concentration for

production was 25% for (maize straw, potato solid wastes

(peels), sugar beet pulp & molasses) (Figs. 13, 14, 15, 16, 17, 18,

19 & 20). Citric acid productivity were obtained by all strains

when using different concentration of the four carbohydrate by-

products when each used alone without any additions after 12

days incubation and the yield enhanced when the fermentation

medium amended with the same concentrations of the mentioned

substrates. Sugar beet pulp and molasses giving the highest yield

by Aspergillus niger strains under the study while maize straw

giving lowest production. The yield on indicator medium and

potato solid wastes almost equal.

It can be concluded that the most potent strains for

production were A1, A4, A5 while A8, A16, A18 & A19 recorded

weak production on indicator medium and the four carbohydrate

by products under the study. Plate (1, 2 & 3) showing production

of citric acid on indicator medium (diameter of clear zone in mm)

of the most potent isolates (A1, A4 & A5), while plate 4, 5, 6 &7

showing clear zone of weak isolates (A8, A16, A18 & A19) (A

particular colony lower the pH of the medium and change the

colour of the indicator medium from purple to yellow).

- 58 -

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


A1 A4 A5

Most potent isolates

5%

10%

15%

20%

25%

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5


A 8 A 16 A 18 A 19

Weak isolates

5%

10%

15%

20%

25%

Fig. (13): Citric acid production (mg/ml) by most potent isolates on basal

medium amended with different concentration of maize straw.

Fig. (14): Citric acid production (mg/ml) by weak isolates on basal medium

amended with different concentration of maize straw.

- 59 -

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


A1 A4 A5


5%

10%

15%

20%

25%

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


A 8 A 16 A 18 A 19

weak isolates

5%

10%

15%

20%

25%


medium amended with different concentration of potato solid

wastes.


amended with different concentration of potato solid wastes.

- 60 -

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


A1 A4 A5


5%

10%

15%

20%

25%

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Citric acid production (mg/m

l)

A 8 A 16 A 18 A 19

Weak isolates

5%

10%

15%

20%

25%

Fig. (17): Citric acid production (mg/ml) by most potent isolates on basal medium

amended with different concentration of sugar beet pulp.


amended with different concentration of sugar beet pulp.

- 61 -

0

0.2

0.4

0.6

0.8

1

1.2


A1 A4 A5


5%

10%

15%

20%

25%

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


A 8 A 16 A 18 A 19

Weak isolates

5%

10%

15%

20%

25%


medium amended with different concentration of molasses.


amended with different concentration of molasses.

- 62 -

Plate (1): A. niger (I1)



Plates (1,2&3) : Most potent isolates of A. niger for citric acid production

on indicator medium after 8 days incubation.

- 63 -

Plate (6) : A. niger (I18) Plate (7) : A. niger (I19)

Plates (4,5,6&7): Weak isolates of A . niger for citric acid production on

indicator medium after 8 days incubation

- 64 -

- 65 -

1. Influence of uv & γγγγ -rays irradiation on the most potent

strains of A. niger for citric acid production.

The parental selected isolates A1, A4 & A5 were exposed to

uv irradiation at different time intervals: i.e. 2, 5, 10, 15, 20, 25 &

30 minutes beside different doses of γ-rays (25, 50, 100 &

200Gy) as a mutagenic agents in a trail for improving the capacity

for citric acid production.

A. UV irradiation: Data recorded in (table 11) showed number of colony

forming unit of selected isolates of A. niger (A1, A4 & A5) on

indicator medium after exposure to uv irradiation. Increasing time

of exposure to uv irradiation led to decrease number of colony

forming units of the selected isolates of Aspergillus niger (A1, A4

& A5). 100% killing of spores was attended within 25 minutes on

indicator medium for A1 & A5 isolates, while within 20 minutes

for A4. Diameter of Clearing zone (mm) for treated (A1, A4 & A5)

after 2, 4 & 6 days incubation are shown in table (12). Maximum

diameter (88mm & 85mm) obtained by T1 & T2 (5min) for A1,

(84-83mm) by T2 (5min) & T1 (10min) for A4, 85mm obtained by

T4 (2 min) & T2 (5min) for A5, all after 6 days incubation.

Production of parental isolates (wild) A1, A4 & A5 on

indicator medium were: 0.96, 0.95 & 0.99mg/ml respectively

(table 13) after 12 days incubation. When comparing that yield of

citric acid with the obtaining resulting isolates (treated with

- 66 -

Table (11): Number of colonyforming units of selected isolates of A.

niger (A1, A4, & A5) on indicator medium after exposure to

uv irradiation.

Dose (min) A1 A4 A5

0 27 23 30

2 5 4 7

5 5 3 5

10 4 3 4

15 2 1 1

20 1 0 1

25 0 0 0

30 0 0 0

- 67 -

Table (12): Screening of treated isolates obtained after

exposure to uv irradiation of selected A. niger

isolates (A1, A4 and A5) for production of citric

acid on indicator medium in terms of diameters

of clearing zone (mm) at different time intervals.

Diameter of clearing zone (mm)

A1 A4 A5

Incubation period (days)

Time of

exposure

(min)

Type

2 4 6 2 4 6 2 4 6

0 P

(wild) 23 54 71 20 45 63 20 50 68

2 T1 28 62 83 20 45 74 23 58 84 T2 25 58 75 23 59 72 25 50 70 T3 26 60 79 25 58 79 25 62 81 T4 27 60 77 25 58 80 23 62 85 T5 24 58 78 - - - 25 56 75 T6 - - - - - - 22 60 80 T7 - - - - - - 25 60 80 5 T1 30 63 88 20 56 76 20 56 80 T2 28 61 85 25 61 84 25 62 85 T3 27 59 76 23 58 80 27 58 74 T4 24 58 75 - - - 22 56 80 T5 27 60 76 - - - 23 56 80

10 T1 25 61 80 26 60 83 21 55 80 T2 26 59 74 25 58 79 24 63 80 T3 27 61 81 25 58 78 22 55 81 T4 24 60 78 - - - 20 58 80

15 T1 27 61 83 25 60 82 20 53 70 T2 25 59 80 - - - - - -

20 T1 26 63 84 - - - 25 60 65 25 0 - - - - - - - - - 30 0 - - - - - - - - -

No of CFU by A1 = 5 at 2 min.: T1 , T2 , T3 , T4 & T5

- 68 -

uv) at that incubation period, maximum production were obtained

by: A4, T2 (5min), followed by A1T1 (5min) & A4 T1 (10min). The

production were 1.78, 1.73 & 1.70 (mg/ml) respectively as shown

from (table 13 & Figs. 21, 22 & 23). Production of parental

isolates A1, A4 & A5 on basal medium with 25% maize straw

were: 0.73, 0.70 & 0.85 (mg/ml) respectively after 12 days

incubation. Maximum yield from the treated (obtained ones) were

: 1.50, 1.45 (mg/ml) by A4 T2 (5 min) & A4 T1 (10min)

respectively with increase 2 fold (table 14 & Figs. 24, 25 & 26).

Yield of citric acid by parental isolates A1, A4, & A5 on

basal medium with 25% potato solid wastes were: 0.94, 0.91 &

0.95 (mg/ml) respectively after 12 days incubation, while

maximum yield by the treated ones were: 1.85, 1.84 & 1.78

(mg/ml) by A1 T1 (5min), A4 T2 (5min) & A1 T3 (5min)

respectively (table 15, Figs. 27, 28 & 29). On basal medium

amended with 25% sugar beet pulp, the production of parental

isolates A1, A4, & A5 after 12 days incubation were: 1.60, 1.50 &

1.51(mg/ml) respectively after 12 days incubation, while

maximum yield by the treated ones after uv treatment reached to

3.16 & 2.82 (mg/ml) with increase 2 fold by A1 T1 (5min) & A1

T5 (5min) respectively (table 16 & Figs. 30, 31 & 32). As for

basal medium amended with 25% molasses the production after

12 days for the parental isolates A1, A4 & A5 were: 1.22, 1.04 &

1.20 (mg/ml) respectively while the yield ranged from (5.00-

5.62mg/ml) for 17 (seventeen) ones obtained (treated with uv

treatment) from the parental A1 after exposure time 2, 5, 15 & 20

minutes with increase reached to 4.5 folds.

- 69 -

Table (13): Citric acid production (mg/ml) of selected

parental isolates of A. niger (A1, A4 and A5) and

obtained isolates (treated) after uv irradiation on

indicator medium. Citric acid production (mg/ml)

A1 A4 A5

Time of exposure

(min) Type

6 days 12 days 6 days 12 days 6 days 12 days

0 P (wild) 0.59±±±±0.06 0.96±±±±0.05 0.57±±±±0.03 0.95±±±±0.06 0.61±±±±0.07 0.99±±±±0.05

2 T1 1.03±±±±0.08 1.43±±±±0.04 0.93±±±±0.04 1.27±±±±0.03 0.43±±±±0.03 0.65±±±±0.08

T2 0.65±±±±0.09 1.14±±±±0.11 0.85±±±±0.08 1.09±±±±0.08 0.82±±±±0.02 1.02±±±±0.10

T3 0.81±±±±0.05 1.29±±±±0.03 1.06±±±±0.02 1.40±±±±0.06 0.70±±±±0.06 0.91±±±±0.04

T4 0.69±±±±0.03 1.28±±±±0.07 1.10±±±±0.06

*

1.50±±±±0.07 1.15±±±±0.08 1.41±±±±0.03

T5 0.76±±±±0.05 1.36±±±±0.05 0 0 0.86±±±±0.07 1.02±±±±0.06

T6 0 0 0 0 0.76±±±±0.04 0.94±±±±0.03

T7 0 0 0 0 0.92±±±±0.05 1.09±±±±0.07

5 T1

1.50±±±±0.07

*

1.73±±±±0.08 0.99±±±±0.11 1.35±±±±0.07 0.50±±±±0.08 0.72±±±±0.08

T2

1.22±±±±0.06

*

1.54±±±±0.08

1.33±±±±0.04

*

1.78±±±±0.07 1.02±±±±0.11 1.18±±±±0.07

T3 1.17±±±±0.11

*

1.52±±±±0.10

1.08±±±±0.13

*

1.45±±±±0.04 0.93±±±±0.09 1.09±±±±0.08

T4 0.80±±±±0.14 1.24±±±±0.12 0 0 0.78±±±±0.03 0.98±±±±0.06

T5

1.20±±±±0.03

*

1.51±±±±0.08 0 0 1.03±±±±0.06 1.40±±±±0.11

10 T1 0.68±±±±0.04 1.09±±±±0.10

1.27±±±±0.09

*

1.70±±±±0.09 0.52±±±±0.09 0.70±±±±0.09

T2

1.12±±±±0.11

*

1.48±±±±0.03

1.03±±±±0.04 1.41±±±±0.08 0.49±±±±0.07 0.71±±±±0.10

T3

1.09±±±±0.08 1.41±±±±0.07

1.05±±±±0.08 1.37±±±±0.06 0.73±±±±0.04 0.90±±±±0.07

T4 1.10±±±±.0.06

*

1.45±±±±0.08 0 0 0.48±±±±0.02 0.70±±±±0.11

15 T1

1.19±±±±0.06

*

1.54±±±±0.07

1.20±±±±0.13

*

1.56±±±±0.10 0.45±±±±0.05 0.67±±±±0.11

T2 0.71±±±±0.04 1.30±±±±0.09 0 0 0 0

20 T1

1.03±±±±0.05

*

1.59±±±±0.06 0 0 0.069±±±±0.07 1.09±±±±0.06

25 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0

* : increase (1.5-2) fold

- 70 -

0.5

0.60.7

0.80.9

1

1.11.2

1.31.4

1.51.6

1.71.8


Time of exposure to u.v light irradiation (min)

p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00

2 5 10 15 20 25 30 0

0.5

0.6

0.7

0.80.9

1

1.11.2

1.3

1.4

1.5

1.6

1.71.8


Time of exposure to u.v light irradiation (m in)


2 5 10 15 20 25 30 0

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5




2 5 10 15 20 25 30 0

Fig. (21): Citric acid production (mg/ml) of selected parental isolate A1 and its

obtained isolates after uv irradiation after 12 days incubation on

indicator medium.



indicator medium.



indicator medium.

A1T

1 A

1T2

A1T

3 A

1T4

A1T

5

- 71 -



obtained isolates (treated) after uv irradiation

growing on basal liquid medium amended with

25% maize straw


A1 A4 A5

Time of exposure

(min) Type


0 P (wild) 0.64±±±±0.06 0.73±±±±0.02 0.61±±±±0.03 0.70±±±±0.06 0.65±±±±0.08 0.85±±±±0.04

2 T1 0.86±±±±0.05 1.05±±±±0.05 0.82±±±±0.08 1.00±±±±0.07 0.70±±±±0.07 0.90±±±±0.07

T2 0.54±±±±0.03 0.90±±±±0.12 0.73±±±±0.04 0.92±±±±0.11 0.63±±±±0.08 0.80±±±±0.09

T3 0.65±±±±0.07 1.00±±±±0.07 0.89±±±±0.06

*

1.11±±±±0.04 0.73±±±±0.04 0.93±±±±0.07

T4 0.61±±±±0.08 0.95±±±±0.11

1.00±±±±0.11

*

1.30±±±±0.08 0.77±±±±0.06 0.95±±±±0.06

T5 0.74±±±±0.09 0.97±±±±0.05 0 0 0.60±±±±0.10 0.78±±±±0.05

T6 0 0 0 0 0.66±±±±0.06 0.88±±±±0.07

T7 0 0 0 0 0.58±±±±0.05 0.79±±±±0.06

5 T1 1.05±±±±0.08

*

1.30±±±±0.06 0.96±±±±0.12

*

1.23±±±±0.05 0.63±±±±0.07 0.85±±±±0.04

T2 0.97±±±±0.08

*

1.18±±±±0.06

1.26±±±±0.03

*

1.50±±±±0.10 0.72±±±±0.03 0.91±±±±0.05

T3 0.94±±±±0.03

*

1.12±±±±0.07

1.02±±±±0.06

*

1.28±±±±0.06 0.70±±±±0.06 0.92±±±±0.03

T4 0.63±±±±0.06 0.82±±±±0.10 0 0 0.67±±±±0.11 0.86±±±±0.09

T5 0.90±±±±0.03 1.09±±±±0.04 0 0 0.61±±±±0.10 0.82±±±±0.06

10 T1 0.70±±±±0.05 0.96±±±±0.07

1.20±±±±0.09

*

1.45±±±±0.04 0.59±±±±0.04 0.78±±±±0.07

T2 0.87±±±±0.09 1.07±±±±0.04 0.90±±±±0.11

*

1.10±±±±0.08 0.82±±±±0.03 0.90±±±±0.10

T3 0.64±±±±0.13 1.00±±±±0.05 0.86±±±±0.03

*

1.08±±±±0.04 0.67±±±±0.07 0.85±±±±0.07

T4 0.91±±±±0.06

*

1.10±±±±0.06 0 0 0.63±±±±0.06 0.82±±±±0.04

15 T1 0.98±±±±0.04

*

1.16±±±±0.10

1.07±±±±0.05 1.31±±±±0.07 0.50±±±±0.04 0.69±±±±0.03

T2 0.82±±±±0.12 1.03±±±±0.04 0 0 0 0

20 T1 0.93±±±±0.05

*

1.22±±±±0.06 0 0 0.45±±±±0.05 0.62±±±±0.07

25 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0


- 72 -

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3Citric acid production (mg/ml)



2 5 10 15 20 25 30 0

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5




2 5 10 15 20 25 30 0

0.5

0.6

0.7

0.8

0.9




2 5 10 15 20 25 30 0


obtained isolates after uv irradiation after 12 days incubation on basal

medium amended with 25% maize straw.







A1T

1 A

1T2

A1T

3 A

1T4

A1T

5

- 73 -



obtained isolates (treated) after uv irradiation

growing on basal liquid medium amended with 25%

potato solid wastes Citric acid production (mg/ml)

A1 A4 A5

Time of exposure

(min) Type


0 P (wild) 0.71±±±±0.05 0.94±±±±0.05 0.74±±±±0.04 0.91±±±±0.07 0.67±±±±0.03 0.95±±±±0.06

2 T1 0.99±±±±0.02 1.30±±±±0.07 0.98±±±±0.09 1.35±±±±0.04 0.70±±±±0.05 0.98±±±±0.04

T2 0.86±±±±0.04 1.17±±±±0.04 0.89±±±±0.08 1.12±±±±0.07 0.73±±±±0.11 0.93±±±±0.06

T3 0.89±±±±0.03 1.21±±±±0.03 1.04±±±±0.03

*

1.47±±±±0.04 0.72±±±±0.03 1.01±±±±0.03

T4 0.81±±±±0.11 1.10±±±±0.08

1.26±±±±0.05

*

1.73±±±±0.08 0.75±±±±0.09 1.06±±±±0.03

T5 0.90±±±±0.08 1.20±±±±0.06 0 0 0.71±±±±0.04 0.95±±±±0.09

T6 0 0 0 0 0.73±±±±0.07 1.00±±±±0.05

T7 0 0 0 0 0.66±±±±0.06 0.91±±±±0.07

5 T1

1.52±±±±0.06

*

1.85±±±±0.04

1.15±±±±0.06

*

1.47±±±±0.03 0.66±±±±0.03 0.95±±±±0.08

T2

1.35±±±±0.04

*

1.72±±±±0.03

1.47±±±±0.08

*

1.84±±±±0.09 0.72±±±±0.09 1.03±±±±0.04

T3

1.43±±±±0.07

*

1.78±±±±0.06

1.26±±±±0.04

*

1.70±±±±0.10 0.71±±±±0.04 0.96±±±±0.02

T4 0.84±±±±0.09 1.12±±±±0.08 0 0 0.70±±±±0.11 0.93±±±±0.09

T5 1.11±±±±0.06

*

1.45±±±±0.08 0 0 0.68±±±±0.05 0.95±±±±0.04

10 T1 1.03±±±±0.05 1.34±±±±0.03

1.41±±±±0.11

*

1.76±±±±0.05 0.63±±±±0.03 0.85±±±±0.06

T2

1.41±±±±0.07

*

1.75±±±±0.09 1.05±±±±0.09

*

1.45±±±±0.12 0.64±±±±0.07 0.88±±±±0.07

T3 0.97±±±±0.03 1.29±±±±0.11 1.03±±±±0.08

*

1.45±±±±0.07 0.70±±±±0.10 0.94±±±±0.08

T4 1.02±±±±0.09 1.34±±±±0.05 0 0 0.70±±±±0.11 0.93±±±±0.09

15 T1

1.35±±±±0.04

*

1.63±±±±0.12

1.28±±±±0.04

*

1.62±±±±0.08 0.66±±±±0.05 0.92±±±±0.10

T2 0.98±±±±0.03 1.31±±±±0.07 0 0 0 0

20 T1

1.20±±±±0.06

*

1.54±±±±0.04 0 0 0.64±±±±0.06 0.90±±±±0.12

25 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0


- 74 -

0.5

0.6

0.70.8

0.9

11.1

1.2

1.3

1.4

1.5

1.61.7

1.8

1.9




2 5 10 15 20 25 30 0

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8




2 5 10 15 20 25 30 0

0.5

0.6

0.7

0.8

0.9

1




2 5 10 15 20 25 30 0

Fig. (27): Citric acid production (mg/ml) of selected parental isolate A1 and its obtained

isolates after uv irradiation after 12 days incubation on basal medium

amended with 25% potato solid wastes.






amended with 25% potato solid wastes

A1T

1 A

1T2

A1T

3 A

1T4

A1T

5

- 75 -

Table (16): Citric acid production (mg/ml) of selected parental

isolates of A. niger (A1, A4 and A5) and obtained isolates

(treated) after uv irradiation growing on basal liquid

medium amended with 25% sugar beet pulp Citric acid production (mg/ml)

A1 A4 A5

Time of exposure

(min) Type


0 P (wild)

1.13±±±±0.03 1.60±±±±0.10 1.13±±±±0.07 1.50±±±±0.09 0.74±±±±0.04 1.51±±±±0.08

2 T1 1.86±±±±0.09 2.16±±±±0.06 1.75±±±±0.04 2.03±±±±0.09 0.66±±±±0.04 1.27±±±±0.04

T2 2.05±±±±0.04 2.32±±±±0.02 1.68±±±±0.08 1.96±±±±0.07 0.64±±±±0.08 1.11±±±±0.07

T3 2.10±±±±0.06 2.36±±±±0.05 1.91±±±±0.06 2.15±±±±0.04 0.78±±±±0.09 1.65±±±±0.03

T4

1.90±±±±0.05 2.18±±±±0.04

2.00±±±±0.05

*

2.30±±±±0.07 0.73±±±±0.05 1.67±±±±0.02

T5 .15±±±±0.05 2.38±±±±0.07 0 0 0.70±±±±0.04 1.47±±±±0.06

T6 0 0 0 0 0.68±±±±0.08 1.37±±±±0.06

T7 0 0 0 0 0.71±±±±0.08 1.49±±±±0.07

5 T1

2.97±±±±0.07

*

3.16±±±±0.04

1.86±±±±0.10 2.23±±±±0.02 0.69±±±±0.09 1.41±±±±0.02

T2

1.96±±±±0.08 2.20±±±±0.06

2.15±±±±0.07

*

2.46±±±±0.06 0.73±±±±0.06 1.51±±±±0.06

T3

2.04±±±±0.02 2.34±±±±0.07

1.98±±±±0.07

*

2.31±±±±0.07 0.65±±±±0.08 1.19±±±±0.07

T4

1.83±±±±0.04 2.12±±±±0.08 0 0 0.72±±±±0.06 1.61±±±±0.05

T5

2.37±±±±0.04

*

2.82±±±±0.05 0 0 0.70±±±±0.05 1.52±±±±0.02

10 T1

2.12±±±±0.07

*

2.50±±±±0.06

2.05±±±±0.09

*

2.42±±±±0.08 0.71±±±±0.06 1.51±±±±0.03

T2

2.17±±±±0.10

*

2.46±±±±0.05

1.88±±±±0.06 2.10±±±±0.06 0.68±±±±0.03 1.39±±±±0.05

T3

2.20±±±±0.09

*

2.52±±±±0.09

1.87±±±±0.10 2.05±±±±0.02 0.70±±±±0.06 1.47±±±±0.11

T4 .79±±±±0.06 2.10±±±±0.12 0 0 0.71±±±±0.04 1.53±±±±0.08

15 T1

1.80±±±±0.10 2.12±±±±0.06

2.00±±±±0.10

*

2.34±±±±0.07 0.69±±±±0.04 1.45±±±±0.04

T2

2.10±±±±0.07

*

2.40±±±±0.09 0 0 00 0

20 T1

2.13±±±±0.08

*

2.58±±±±0.03 0 0 0.67±±±±0.06 1.33±±±±0.07

25 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0


- 76 -

25 0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9




2 5 10 15 20 30 0

0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9




2 5 10 15 20 25 30 0

20 0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8




2 5 10 15 25 30 0



amended with 25% sugar beet pulp.






basal medium amended with 25% sugar beet pulp.

A1T

1 A

1T2

A1T

3 A

1T4

A1T

5

- 77 -

Maximum yield obtained with A1 T4 (2min), A1 T1 & T3 (5

min), A1 T2 & T4 (10min), the production was: 5.5, 5.6, 5.5, 5.5

and 5.56 respectively, (table 17& Fig. 33). As for A4, 11 (eleven)

obtained ones recorded 2 fold increase in production when

compared to the parental isolate (non-treated) Fig. (34). While A5,

9 (min) derivatives recorded 2 fold increase (table 17 & Fig. 35).

B. γγγγ-rays:

(Table 18) showed number of colony-forming units of

selected isolates of Aspergillus niger (A1, A4 & A5) on indicator

medium after exposure to γ-rays. Decrease of colonies was shown

in (table 19) at doses under the study (25, 50, 100 & 200Gy)

for the three isolates. Data in table (20) revealed increase 2 fold

on indicator medium for the treated ones after γ-rays treatment

after 12 days incubation in derivatives A1: (T1 50Gy; T1-T2 –T3 &

T4 100Gy; T1 & T2200 Gy) (Fig. 36). A4 (Fig. 37) (T1, T2 & T3,

100Gy) A5. (Fig. 38) (T1 & T2 100Gy; T1 & T2 200Gy).

Slight increase in citric acid production in few obtained

ones on based medium with 25% maize straw when compared

with parental isolates (untreated with γ-rays) (table 21 & Figs.

(39, 40 & 41) when the basal medium amended with 25% potato

solid wastes, maximum citric acid production after 12 days

incubation and γ-rays treatment were: 1.80, 1.65, 1.60 & 1.63

(mg/ml) by A1 (T1, T2, T3 & T4, after exposed to 100Gy)

- 78 -

Table (17): Citric acid production (mg/ml) of selected parental isolates

of A. niger (A1, A4 and A5) and obtained isolates (treated) after uv

irradiation growing on basal liquid medium amended with 25%

molasses.


A1 A4 A5

Time of exposure

(min) Type


0 P (wild) 1.12±±±±0.09 1.22±±±±0.10 0.95±±±±0.03 1.04±±±±0.05 1.02±±±±0.09 1.20±±±±0.07

2 T1

4.57±±±±0.07

***

5.38±±±±0.08 2.21±±±±0.10

*

2.62±±±±0.04 1.59±±±±0.04 2.14±±±±0.05

T2

4.40±±±±0.11

***

5.30±±±±0.09 1.70±±±±0.09

*

2.07±±±±0.09 1.79±±±±0.07 2.03±±±±0.04

T3

4.42±±±±0.06

***

5.30±±±±0.12 2.34±±±±0.07

*

2.80±±±±0.02 2.25±±±±0.06

*

3.20±±±±0.03

T4

4.72±±±±0.09

****

5.50±±±±0.04

2.50±±±±0.06

*

2.91±±±±0.05

2.84±±±±0.03

**

3.70±±±±0.03

T5

4.21±±±±0.08

***

5.12±±±±0.06 0 0 2.01±±±±0.04

*

2.72±±±±0.07

T6 0 0 0 0 1.52±±±±0.09

*

2.40±±±±0.08

T7 0 0 0 0 1.72±±±±0.12

*

2.30±±±±0.06

5 T1 4.90±±±±0.04

**** 5.62±±±±0.06 2.30±±±±0.11

* 2.71±±±±0.04 1.52±±±±0.07 2.17±±±±0.06

T2 4.50±±±±0.05

*** 5.36±±±±0.07

2.71±±±±0.08

* 3.05±±±±0.06 1.83±±±±0.05

* 3.12±±±±0.09

T3 4.70±±±±0.07

**** 5.50±±±±0.12

2.49±±±±0.04

* 2.90±±±±0.07 0.97±±±±0.03 1.58±±±±0.10

T4 4.16±±±±0.08

*** 5.02±±±±0.04 0 0 1.62±±±±0.13 2.35±±±±0.07

T5 4.23±±±±0.12

*** 5.12±±±±0.09 0 0 2.14±±±±0.07

* 2.40±±±±0.04

10 T1 4.66±±±±0.10

*** 5.44±±±±0.03

2.60±±±±0.03

* 2.98±±±±0.07 1.97±±±±0.07

* 2.68±±±±0.06

T2 4.44±±±±0.06

**** 5.50±±±±0.08 2.30±±±±0.13

* 2.78±±±±0.09 1.70±±±±0.08

1.98±±±±0.08

T3 4.30±±±±0.07

** 5.15±±±±0.13 2.27±±±±0.06

* 2.70±±±±0.05 1.91±±±±0.08

* 2.52±±±±0.09

T4

4.81±±±±0.05

****

5.56±±±±0.07 0 0 1.60±±±±0.07 2.17±±±±0.05

15 T1 4.59±±±±0.09

*** 5.39±±±±0.10

2.50±±±±0.08

* 2.84±±±±0.07 1.79±±±±0.04 2.30±±±±0.03

T2 4.48±±±±0.03

*** 5.32±±±±0.05 0 0 0 0

20 T1 4.45±±±±0.06

*** 5.34±±±±0.05 0 0 1.66±±±±0.03 2.21±±±±0.09

25 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0

****:increase (4.5fold)***:increase(4fold)**:increase (3fold)*increase (2 fold)

- 79 -

0.1

0.50.9

1.31.7

2.12.5

2.93.3

3.74.1

4.54.9

5.35.7




2 5 10 15 20 25 30 0

0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9




2 5 10 15 20 25 30 0

0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9

3.3

3.7




2 5 10 15 20 25 30 0










A1T

1 A

1T2

A1T

3 A

1T4

A1T

5

- 80 -

Table (18): Number of colony-forming units of selected

isolates of A. niger (A1, A4 and A5) on indicator

medium after exposure to gamma rays.

Dose (Gy) A1 A4 A5

0

25

50

100

200

27

1

3

4

2

23

1

2

3

1

30

1

1

2

2

- 81 -

Table (19): Screening of treated isolates obtained after gamma rays

irradiation of selected A. niger isolates (A1, A4 and A5) for

production of citric acid and obtained isolates on indicator

medium in terms of diameter of clearing zone (mm) at

different time intervals.


A1 A4 A5

Days days days Dose

(Gy) Type 2 4 6 2 4 6 2 4 6

0 P

(wild) 23 54 71 20 45 63 20 50 68

25 T1 24 55 69 22 50 64 21 54 68

50 T1 25 57 72 23 51 67 22 55 70

T2 21 57 71 24 53 69 0 0 0

T3 21 50 70 0 0 0 0 0 0

100 T1 24 56 74 23 54 70 25 58 73

T2 24 57 72 24 55 71 24 57 73

T3 22 56 70 22 53 70 0 0 0

T4 23 56 71 0 0 0 0 0 0

200 T1 23 55 72 23 51 65 22 54 70

T2 22 56 74 0 0 0 21 53 72

- 82 -



obtained isolates (treated) after gamma rays

irradiation on indicator liquid medium Citric acid production (mg/ml)

A1 A4 A5 Dose (Gy.)

Type


0 P

(wild) 0.59±±±±0.09 0.96±±±±0.10 0.57±±±±0.08 0.95±±±±0.04 0.61±±±±0.05 0.99±±±±0.07

25 T1 0.55±±±±0.06 1.40±±±±0.011 0.60±±±±0.06 1.40±±±±0.05 0.63±±±±0.08 1.80±±±±0.04

50 T1 1.00±±±±0.07

*

1.90±±±±0.06 0.63±±±±0.09 1.76±±±±0.04

1.23±±±±0.06 1.87±±±±0.09

T2 0.79±±±±0.09 1.80±±±±0.05 0.72±±±±0.03 1.69±±±±0.08 0 0

T3 0.74±±±±0.05 1.74±±±±0.07 0 0 0 0

100 T1

1.58±±±±0.11

*

2.15±±±±0.04

1.12±±±±0.12

*

1.82±±±±0.09

1.54±±±±0.09

*

2.10±±±±0.03

T2

1.44±±±±0.05

*

2.12±±±±0.12

1.22±±±±0.04

*

1.87±±±±0.06

1.59±±±±0.04

*

2.04±±±±0.04

T3

1.20±±±±0.07

*

2.00±±±±0.08

1.14±±±±0.07

1.79±±±±0.06 0 0

T4

1.26±±±±0.08

*

1.96±±±±0.03 0 0 0 0

200 T1 1.39±±±±0.06

*

2.10±±±±0.07 0.98±±±±0.06 1.40±±±±0.03

1.25±±±±0.11 1.95±±±±0.10

T2

1.49±±±±0.03

*

2.15±±±±0.08 0 0

1.39±±±±0.08

*

1.98±±±±0.06

* : increase (2 fold)

- 83 -

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1


Dose of gamma rays (Gy)

p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1



p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.10.20.30.40.50.60.70.80.91

1.11.21.31.41.51.61.71.81.92

2.1


l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0


obtained isolates after gamma rays irradiation after 12 days








- 84 -

Table (21): Citric acids production (mg/ml) of selected




amended with 25% maize straw


A1 A4 A5 Dose (Gy.)

Type


0 P (wild) 0.64±±±±0.11 0.73±±±±0.09 0.61±±±±0.10 0.70±±±±0.13 0.65±±±±0.06 0.85±±±±0.03

25 T1 0.63±±±±0.08 0.70±±±±0.06 0.67±±±±0.05 0.71±±±±0.09 0.64±±±±0.05 0.76±±±±0.09

50 T1 0.75±±±±0.06 0.83±±±±0.04 0.70±±±±0.09 0.75±±±±0.04 0.70±±±±0.09 0.84±±±±0.08

T2 0.72±±±±0.05 0.88±±±±0.11 0.69±±±±0.07 0.77±±±±0.05 0 0

T3 0.70±±±±0.07 0.79±±±±0.10 0 0 0 0

100 T1 0.94±±±±0.03 1.10±±±±0.03 0.70±±±±0.05 0.80±±±±0.06 0.90±±±±0.11 1.03±±±±0.05

T2 0.78±±±±0.06 1.00±±±±0.06 0.76±±±±0.08 0.84±±±±0.07 0.95±±±±0.04 1.09±±±±0.07

T3 0.70±±±±0.02 0.94±±±±0.07 0.71±±±±0.04 0.79±±±±0.08 0 0

T4 0.76±±±±0.10 0.96±±±±0.09 0 0 0 0

200 T1 0.80±±±±0.09 1.02±±±±0.13 0.68±±±±0.03 0.75±±±±0.06 0.72±±±±0.06 0.89±±±±0.12

T2 0.90±±±±0.08 1.05±±±±0.06 0 0 0.80±±±±0.07 0.94±±±±0.03

- 85 -

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1


l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1


l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0


isolates after gamma rays irradiation after 12 days incubation on basal








- 86 -

with increase 1.5 fold (table 22) (Fig. 42), no increase in

production obtained in the derivatives for A4 & A5 after all

doses, of γ-rays under the study (Figs. 43, 44). As for sugar beet

pulp, slight increase in citric acid production for some obtained

ones on that carbohydrate by-product (table 23, Figs 45, 46 & 47)

when compared to the parental isolates (untreated with γ-rays).

On basal medium amended with 25% molasses, increase in

production for ten (10) obtained ones for A1 after treatment with

γ- rays. 2 fold recorded after 12 days incubation by A1 (T1 100Gy)

& 1.5 fold for (T1) 25Gy; (T1, T2, T3) 50Gy; (T2, T3, T4) 100Gy &

(T1, T2) 200Gy (tables 24 & 48). Also increases in production

reached to 1.5 folds recorded in the table for some obtained ones

in this carbohydrate by-product by A4 & A5 strains (Figs. 49 &

50).

From the data obtained in part II it can be concluded that uv

treatment affect positively citric acid production by selected

isolates of A. niger growing on different carbohydrate by

products. Molasses was the best carbohydrate by product for citric

acid production when added at 25% conc. to the basal medium

after 12 days incubation (increase reached to 5 fold in some

obtained ones after uv treatment from the parental isolate, A1 as

mentioned followed by sugar beet pulp, while the lowest yield

recorded with maize straw. Production by treated isolates (after

uv treatment) growing on indicator medium & basal medium with

potato solid wastes almost equal, both better than with maize

straw but lowest than molasses and sugar beet pulp. As for

γ-rays, increase in

- 87 -







A1 A4 A5 Dose (Gy.)

Type


0 P (wild) 0.71±±±±0.08 0.94±±±±0.06 0.74±±±±0.03 0.91±±±±0.08 0.67±±±±0.04 0.95±±±±0.03

25 T1 0.68±±±±0.07 0.90±±±±0.04 0.71±±±±0.08 0.94±±±±0.09 0.70±±±±0.06 0.97±±±±0.10

50 T1 0.89±±±±0.10 0.96±±±±0.06 0.74±±±±0.07 0.96±±±±0.04 0.77±±±±0.05 1.00±±±±0.03

T2 0.76±±±±0.03 1.03±±±±0.05 0.76±±±±0.05 0.98±±±±0.06 0 0

T3 0.74±±±±0.07 1.00±±±±0.06 0 0 0 0

100 T1 1.00±±±±0.09

*

1.80±±±±0.07 0.80±±±±0.04 1.10±±±±0.10 0.93±±±±0.09 1.14±±±±0.12

T2 0.86±±±±0.06

*

1.65±±±±0.08 0.82±±±±0.06 1.08±±±±0.09 1.00±±±±0.03 1.20±±±±0.10

T3 0.76±±±±0.08

*

1.60±±±±0.09 0.79±±±±0.04 1.05±±±±0.06 0 0

T4 0.80±±±±0.06

*

1.63±±±±0.04 0 0 0 0

200 T1 0.85±±±±0.12 1.15±±±±0.03 0.76±±±±0.04 1.00±±±±0.07 0.80±±±±0.04 0.99±±±±0.07

T2 0.96±±±±0.10 1.20±±±±0.09 0 0 0.84±±±±0.06 1.05±±±±0.08

* : increase (1.5 fold)

- 88 -

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

Citric acid pro

duction (mg/m

l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.10.20.30.40.50.60.70.80.91

1.11.21.31.41.51.61.71.8


l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1


l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0



medium amended with 25% potato solid wastes.







- 89 -







A1 A4 A5 Dose (Gy.)

Type


0 P

(wild) 1.13±±±±0.07 1.60±±±±0.06 1.13±±±±0.09 1.50±±±±0.04 0.74±±±±0.09 1.51±±±±0.13

25 T1 0.94±±±±0.11 1.20±±±±0.10 0.72±±±±0.06 1.00±±±±0.07 0.76±±±±0.05 1.15±±±±0.06

50 T1 1.20±±±±0.10 1.56±±±±0.13 0.80±±±±0.07 1.07±±±±0.08 1.13±±±±0.04 1.60±±±±0.07

T2 1.16±±±±0.08 1.67±±±±0.04 1.09±±±±0.04 1.30±±±±0.12 0 0

T3 1.00±±±±0.07 1.55±±±±0.06 0 0 0 0

100 T1 1.60±±±±0.06 2.10±±±±0.09 1.00±±±±0.03 1.76±±±±0.14 1.46±±±±0.08 1.94±±±±0.09

T2 1.34±±±±0.03 1.87±±±±0.06 1.04±±±±0.08 1.70±±±±0.09 1.42±±±±0.07 1.81±±±±0.03

T3 1.11±±±±0.07 1.67±±±±0.07 1.00±±±±0.07 1.65±±±±0.06 0 0

T4 1.25±±±±0.06 1.73±±±±0.04 0 0 0 0

200 T1 1.40±±±±0.08 1.85±±±±0.02 0.95±±±±0.06 1.35±±±±0.04 1.00±±±±0.05 1.60±±±±0.04

T2 1.46±±±±0.05 1.99±±±±0.07 0 0 1.17±±±±0.04 1.67±±±±0.08

- 90 -

0.10.20.30.40.50.60.70.80.91

1.11.21.31.41.51.61.71.81.92

2.1



p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.10.20.30.40.50.60.70.80.91

1.11.21.31.41.51.61.71.8



p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.10.20.30.40.50.60.70.80.91

1.11.21.31.41.51.61.71.81.9



p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0



incubation on basal medium amended with 25% sugar beet pulp.







- 91 -





amended with 25% molasses.


A1 A4 A5 Dose (Gy.)

Type


0 P

(wild) 1.09±±±±0.04 1.20±±±±0.07 0.92±±±±0.09 1.00±±±±0.08 1.00±±±±0.06 1.17±±±±0.10

25 T1 1.00±±±±0.08

*

1.88±±±±0.06 0.95±±±±0.03

*

1.5±±±±0.10 0.98±±±±0.04

*

2.03±±±±0.08

50 T1 1.20±±±±0.06

*

2.21±±±±0.05 0.99±±±±0.04

*

1.87±±±±0.05 1.25±±±±0.06

*

2.07±±±±0.05

T2 1.12±±±±0.04

*

2.08±±±±0.06 1.01±±±±0.09

*

1.94±±±±0.03 0 0

T3 1.03±±±±0.03

*

1.95±±±±0.04 0 0 0 0

100 T1 1.60±±±±0.09

**

2.40±±±±0.03 1.08±±±±0.03 1.75±±±±0.07

1.65±±±±0.03

*

2.12±±±±0.07

T2 1.44±±±±0.8

*

2.25±±±±0.06 1.12±±±±0.05

*

1.72±±±±0.04

1.61±±±±0.08

*

2.10±±±±0.04

T3 1.26±±±±0.07

*

2.15±±±±0.07 1.10±±±±0.07

*

1.80±±±±0.06 0 0

T4 1.34±±±±0.04

*

2.25±±±±0.09 0 0 0 0

200 T1 1.43±±±±0.03

*

2.15±±±±0.10 0.97±±±±0.06

*

1.70±±±±0.03 1.07±±±±0.09

*

1.97±±±±0.03

T2 1.48±±±±0.09

*

2.20±±±±0.08 0 0 1.20±±±±0.10

*

2.08±±±±0.09

* : increase (1.5 fold) * * : increase (2 fold)

- 92 -

0.10.30.50.70.91.11.31.51.71.92.12.32.5

Citric acid

production (mg/ml)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T225 50 100 200 0

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

2.5



p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0

0.10.20.30.40.50.60.70.80.91

1.11.21.31.41.51.61.71.81.92

2.1


l)


p 0

T1 0

T1 T2

T3 0

T1 T2

T3 T4

0 T1

T2

25 50 100 200 0


obtained isolates after gamma rays irradiation after 12 days incubation on

basal medium amended with 25% molasses.







- 93 -

production (2 fold) with some obtained ones on molasses only.

UV treatment was more effective than γ-rays in producing

number of obtained isolates of A. niger yielding more production

of citric acid than the parental strains.

2. Influence of uv irradiation on weak isolates of A. niger for

citric acid production:

Table (25) showed number of colony forming units of weak

isolates of A. niger (A8, A16, A18, & A19) on indicator medium

after uv treatment. Decrease of colony forming units were

obtained after 10 & 15 minutes from exposure. 100% killing of

spores after 20 minutes of exposure for A16 while within 25

minutes for A8, A18 & A19.

Production of these parental isolates after 12 days

incubation on that medium were: 0.74, 0.70, 0.69 & 0.60 (mg/ml)

respectively. Interestingly, increase of production fold reached to

5, 6, 7, 8 & 9 folds as recorded in (table 27) when comparing the

production with the wild (parental) isolates after uv irradiation.

Maximum production by A8 were obtained by T1, T2, T3, T4, & T5

(10 minutes), the production were 3.6, 3.5, 3.43, 3.04 & 3.15

(mg/ml) respectively (Fig. 51) with increase of 4.5 folds. Also 10

minutes achieved maximum production for A16, obtained by T1,

T2, T3, T4 & T5 as shown from the table (27) and Fig.(52), the

production after 12 days were: 5.2, 5.15, 4.2, 4.87 & 4.13 (mg/ml)

- 94 -

respectively with increase 6 & 7 folds. Again 10 minutes achieved

maximum production for A18 by T1, T2, T3, T4 & T5 also T1

(15min), the production were: 5.2, 5.17, 4.65, 4.8, 4.0 & 4.2

(mg/ml) respectively with 6-7 fold increase (Fig. 53). Production

reached to 8.5 fold increase with A19, T1 & T2 (10 minutes),

production were: 5.35 & 5.25 (mg/ml) respectively (Fig. 54).

In conclusion, pronounced increase in citric acid production

recorded in this study by weak isolates when exposed to U.V

treatment growing on indicator medium, when compared the

derivatives ones with the parental strains. A19, T1 (10 minutes)

was chosen for PCR studies, 9 fold increase was obtained after

12 days incubation on indicator medium as compared to the

parental isolate. Table (28) showed citric acid production of this

isolate on different carbohydrate by-products in 12 days

incubation. Molasses recorded the highest yield for this obtained

isolate followed by sugar beet pulp and potato solid wastes, maize

straw recorded the lowest the lowest production (Fig. 55 & 56).

Plate 7- showed citric acid production of parental isolate A19

(clear zone) on indicator medium after 12 days incubation while

plate 8 showed derivative isolate of A19 after uv treatment

(10mim) after 6 days incubation (A19 , T1).

- 95 -

Table (25): Number of colony-forming units of weak isolates

of A. niger (I8, I16, I18 and I19) on indicator

medium after uv irradiation.

Dose

(min)

A8 A16 A18 A19

0

2

5

10

15

20

25

30

13

11

10

5

3

1

0

0

11

10

10

4

1

0

0

0

12

10

9

4

2

1

0

0

10

8

8

3

1

1

0

0

- 96 -

Table (26): Screening of treated isolates obtained after uv

irradiation of parental weak isolates of A. niger

(A8, A16, A18 and A19) for production of citric acid

on indicator medium in terms of diameter of

clearing zone (mm) at different time intervals.


A8 A16 A18 A19

days days days days

Dose (min)

Type

2 4 6 2 4 6 2 4 6 2 4 6

0 P

(Wild) 20 30 34 20 22 25 20 25 25 10 17 20

2 T1 21 32 36 21 25 29 22 26 30 15 20 23

5 T1 22 34 38 21 27 30 21 29 32 19 22 25

10 T1 25 60 85 24 62 87 24 64 88 28 62 90

T2 27 56 75 28 64 87 23 61 87 25 60 90

T3 25 58 75 25 60 79 22 55 80 27 58 82

T4 23 51 70 24 64 84 22 58 83 24 61 81

T5 23 54 72 24 53 76 21 51 75 25 60 80

15 T1 22 50 58 22 50 75 21 54 77 23 67 83

T2 20 32 50 21 47 68 22 50 70 22 54 72

T3 21 30 45 22 49 77 21 48 71 22 57 75

20 T1 20 29 38 0 0 0 21 45 60 21 51 70

25 0 0 0 0 0 0 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0 0 0 0 0 0 0

- 97 -

Table (27): Citric acid production by parental weak isolates

(A8, A16, A18 and A19) of A. niger and obtained isolates after uv

irradionlion on indicator medium at different time intervals.

***** : increase (8.5 fold) **** : increase (8 fold) *** : increase (7 fold) ** : increase (6 fold) * : increase (5 fold)


A8 A16 A18 A19

Days days days days

Time of exposure

to u.v. (min)

Type

6 12 6 12 6 12 6 12

0 P

(Wild) 0.41±±±±0.05 0.74±±±±0.09 0.35±±±±0.04 0.70±±±±0.08 0.38±±±±0.07 0.69±±±±0.03 0.28±±±±0.04 0.60±±±±0.08

2 T1 0.53±±±±0.07 0.80±±±±0.09 0.50±±±±0.08 0.73±±±±0.04 0.52±±±±0.07 0.75±±±±0.06 0.40±±±±0.07 0.83±±±±0.04

5 T1 0.74±±±±0.04 0.83±±±±0.09 0.61±±±±0.07 0.80±±±±0.03 0.65±±±±0.04 0.90±±±±0.07 0.42±±±±0.07 0.90±±±±0.03

10 T1

2.70±±±±0.05

*

3.60±±±±0.08

2.57±±±±0.06

***

5.20±±±±0.05

0.65±±±±0.03

***

5.20±±±±0.04

2.70±±±±0.04

*****

5.35±±±±0.07

T2

2.40±±±±0.08

3.50±±±±0.06

2.51±±±±0.05

***

5.15±±±±0.06

2.60±±±±0.07

***

5.17±±±±0.06

2.65±±±±0.05

*****

5.25±±±±0.05

T3

2.32±±±±0.09

3.43±±±±0.05

2.25±±±±0.06

**

4.20±±±±0.07

2.28±±±±0.07

**

4.65±±±±0.03

2.20±±±±0.06

***

4.80±±±±0.04

T4

1.85±±±±0.03 3.04±±±±0.04

2.38±±±±0.04

***

4.87±±±±0.04

2.40±±±±0.08

***

4.80±±±±0.07

2.15±±±±0.07

***

4.60±±±±0.03

T5

1.93±±±±0.06 3.15±±±±0.08

2.12±±±±0.09

**

4.13±±±±0.08

2.17±±±±0.06

*

4.00±±±±0.08

2.12±±±±0.08

***

4.45±±±±0.07

15 T1 1.70±±±±0.07 2.50±±±±0.09

2.10±±±±0.09

**

3.90±±±±0.09

2.20±±±±0.05

**

4.12±±±±0.06

2.29±±±±0.11

****

4.78±±±±0.06

T2 1.60±±±±0.04 2.03±±±±0.04

1.86±±±±0.08

*

3.41±±±±0.03

1.92±±±±0.11

*

3.50±±±±0.07

2.02±±±±0.14

**

3.65±±±±0.09

T3 1.52±±±±0.04 1.95±±±±0.10

1.93±±±±0.07

*

3.52±±±±0.06

1.99±±±±0.10

3.23±±±±0.06

2.09±±±±0.07

**

3.86±±±±0.04

20 T1 1.44±±±±0.03 1.75±±±±0.05 0 0

1.88±±±±0.02 3.00±±±±0.05

1.98±±±±0.05

**

3.50±±±±0.06

25 0 0 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0 0 0

- 98 -

0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9

3.3

3.7


l)


p 0

T1 0

T1 0

T1 T2

T3 T4

T5 0

T1 T2

T3 0

T1 0

0 0

0

2 5 10 15 20 25 30 0

0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9

3.3

3.7

4.1

4.5

4.9

5.3



p 0

T1 0

T1 0

T1 T2

T3 T4

T5 0

T1 T2

T3 0

T1 0

0 0

0

2 5 10 15 20 25 30 0

0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9

3.3

3.7

4.1

4.5

4.9

5.3


l)


p 0

T1 0

T1 0

T1 T2

T3 T4

T5 0

T1 T2

T3 0

T1 0

0 0

0

2 5 10 15 20 25 30 0

Fig. (51): Citric acid production (mg/ml) of parental weak isolate A8 and its

obtained isolates after exposure to u.v. irradiation after 12 days

incubation on indicator medium







- 99 -

0.1

0.5

0.9

1.3

1.7

2.1

2.5

2.9

3.3

3.7

4.1

4.5

4.9

5.3


l)


p 0

T1 0

T1 0

T1 T2

T3 T4

T5 0

T1 T2

T3 0

T1 0

0 0

0

2 5 10 15 20 25 30 0

0

1

2

3

4

5

6

7


Maize straw Potato solid wastes Sugar beet pulp Molasses

Carbohydrarte by products

0

5

10

15

20

25

30

35

40

45

Substrate consumed (%)

Maize straw Potato solid wastes Sugar beet pulp Molasses

Carbohydrarte by products

Fig. (54): Citric acid production (mg/ml) of parental weak isolate A19 and its obtained

isolates after uv irradiation after 12 days incubation on indicator medium

.

Fig. (55): Citric acid production (mg/ml) by A19 (T1 u.v.10) of A. niger on different

carbohydrate by products after 12 days incubation.

Fig. (56): Substrate consumed (%) by A19 (T1 u.v.10) of A. niger on different

carbohydrate by products after 12 days incubation.

- 100 -

Table (28): Citric acid production (mg/ml) by A19 (T1 u.v.10)

of A. niger on different carbohydrate by

products after 12 days incubation.

Carbohydrate

by products Citric acid mg/ml Substrate consumed (%)

Maize straw 2.05 0.6

Potato solid wastes 2.83 2.5

Sugar beet pulp 3.80 15

Molasses 6.10 41.06

Weight of substrate before fermentation - Weight of substrate after fermentation Substrate

consumed % =

Weight of substrate before fermentation X 100

- 101 -

Plate (7): Wild (Parental isolate) of A.niger (I 19) on indicator medium

after 8 days incubation.

(Obtained isolate)

Plate (8): Derivative isolate of A. niger (I19) after exposure to U.V. light

irradiation (10 minutes) after 6 days incubation on indicator

medium (A19 T1).

- 102 -

- 103 -

From the previous study and the obtained results,

Aspergillus niger A1 (potent strain) and A1T1 (5 min) in addition

to A19 (weak strain) and A19 T1 (10 min) were chosen for PCR and

protein electrophoresis studies.

RAPD analysis by using PCR has been used successfully to

indentify genetic variability on the molecular level between wild

strain of Aspergillus niger (A1 & A19) and its treated isolates with

uv irradiation at 5 min &10 min respectively i.e. (A1 T1) & (A19

T1). Ten arbitrary primers were used for PCR amplification of

total DNAs of the (A1&A1T15min). Figures (57-61) showed the

amplified product of bands, and total number of the amplified

RAPD bands produced by the 10 arbitrary primers that is present

in table (29).

As shown from the mentioned table and figures, RAPD

analysis revealed that three primers (OPB-10, OPB-08 & OPA-

15) showed no polymorphism among two strains (A1 and A1T1).

On the other hand, it was found that seven primers (OPO-14,

OPO-02, OPA-05, OPA-04, OPA-18, OPC-14 & OPB-05)

showed variations in number of bands between the two mentioned

strains. These variations come from the mutagenic effect of uv

irradiation on the Aspergillus niger .

- 104 -

Table (29): Number of amplified DNA bands scored for the

wild strain A. niger A1 (potent isolate) and its obtained isolate

(A1 T1 5min) by uv irradiation.

No. Primers

Wild

strain

(A1)

Obtained

isolate (A1T1

5min)

No of

total

bands

No of

shared

bands

1

2

3

4

5

6

7

8

9

10

OPO-14

OPO-02

OPB-10

OPB-08

OPA-05

OPA-04

OPA-15

OPA-18

OPC-14

OPB-05

4

4

2

4

10

1

4

3

2

5

3

2

2

4

8

4

4

4

1

3

7

6

4

8

18

5

8

7

3

8

2

2

2

4

8

1

4

3

1

3

Data in table (30) indicated the unique positive and/or

negative marker and their molecular weight (size) generated by

RAPD analysis for the two strains (A1& A1,T1 5 min). Data

revealed that five positive unique markers identified the

mutantA1T1 5min, amplified by OPO-14 primer at molecular

weight of 4245 bp (base pairs) OPA-18 primer, at molecular

weight of 811bp, and OPA-04 primer at molecular weight 572,

219 and 135bp, these five bands found only on A1T1.

- 105 -

In the wild isolate A1 nine unique markers amplified by

OPO-02 primer at molecular weight of 2115 and 811bp, OPO-14

primer at molecular weight of 573 & 325bp, OPC-14 primer at

molecular weight of 548bp, OPA-05 primer at molecular weight

604 & 480 bp and OPB-05 primer at molecular weight 453 &

272bp.

Table (30): Positive and negative unique RAPD markers and

their molecular weight of the two strains of A.

niger (A1 & A1 T1 5 min).

Primers Approx band size in bp A1 A1 T1

OPO-02 2115

811

1

1

0

0

OPO-14

4245

573

325

0

1

1

1

0

0

OPC-14 548 1 0

OPA-05 604

480

1

1

0

0

OPB-05 453

272

1

1

0

0

OPA-18 811 0 1

OPA-04

572

219

135

0

0

0

1

1

1 Presence of a band in a genotype = 1 Absence of the shared band in the other genotype = 0

- 106 -

Fig. (57): RAPD banding patterns amplified for A. niger (A1) and its

obtained isolate(A1T15min) using primers(OPO-14&OPO-02)

M (DNA marker) = 5Kb ladder.

T1 A1 M T1 A1

OPB-10 OPB-08


obtained isolate (A1T15min) using primers(OPB-10&OPB-08)

M (DNA marker) = 1Kb ladder.

- 107 -

A1 T1 M

OPA-05

Fig (59): RAPD banding patterns amplified for A. niger (A1) and its

obtained isolate (A1 T1 5min) using primer (OPA-05) M

(DNA marker) = 1 Kb ladder.

A1 T1 M A1 T1 M A1 T1

OPA-18 OPA-15 OPA-04


obtained isolate (A1T1 5min) using primers (OPA-04, OPA-15

& OPA-18) M (DNA marker) = 1 Kb ladder.

- 108 -

A1 T1 M A1 T1

OPB-05 OPC-14

Fig. (61): RAPD banding patterns amplified for A. niger (A1) & its

obtained isolate (A1 T1 5min) using primers (OPC-14 & OPB-

05) M (DNA marker) = 1kb ladder.

Figures (62-71) showed the amplified products of bands and total number of the amplified RAPD bands produced by the 10 random primers for A. niger wild strain A19 (weak isolate) & its obtained isolate after uv irradiation (A19T1 10min) and presented in table (31). RAPD analysis revealed that 3 out of 10 primers OPC-20, OPO-04 & OPB-05 showed similarities in bands between the two strain (wild & obtained isolate). On the other hand, it was found that the amplified product by other seven primers (OPG-05, OPB-15, OPB-06, OPA-20, OPH-15, OPC-10 & OPB-07) showed difference in number of bands between the genotypes A19 & A19 T1 (10min). These variations come from the mutagenic effect of uv irradiation on A. niger.

- 109 -

Table (31): Number of amplified DNA bands scored for the

wild strain of A. niger A19 and its obtained isolate

(A19T1 10 min) by uv irradiation.

No. Primers Wild strain

(A19)

Obtained

isolate (A19T1

10min)

No of

total

bands

No of

shared

bands

1

2

3

4

5

6

7

8

9

10

OPG-05

OPH-15

OPC-10

OPC-20

OPA-20

OPO-04

OPB-15

OPB-05

OPB-07

OPB-06

2

5

7

8

4

6

4

6

5

4

3

8

6

8

6

6

3

6

6

3

5

13

13

16

10

12

7

12

11

7

2

1

6

8

4

6

3

6

0

3

- 110 -

M A19 T1

OPG-05


obtained isolate (A19 T1 10 min) using primer OPG-05 M

(DNA marker) = 1kb ladder.

- 111 -

M A19 T1

OPH-15


obtained isolate (A19 T1 10min) using primer OPH-15 M

(DNA marker) = 100 base pairs ladder.

A19 T1 M

OPC-10

Fig. (64): RAPD banding patterns amplified A. niger (A19) and its

obtained isolate (A19 T1 10min) using primer OPC-10 M

(DNA marker) = 1Kb ladder.

- 112 -

T1 A19 M

OPC-20


obtained isolated (A19 T1 10min) using primer OPC -20 M

(DNA marker) = 1 kb ladder.

A19 T1 M

OPA-20

Fig. (66): RAPD banding patterns amplified for A niger (A19) and its

obtained isolate (A19 T1 10 min) using primer OPA-20 M

(DNA marker) = 1kb

- 113 -


obtained isolate (A19 T1 10min) using primer OPO-04 M

(DNA marker) = 1Kb.

A19 T1 M

OPB-15


obtained isolate (A19 T1 10min) using primer OPB-15 M

(DNA marker) = 1kb ladder.

- 114 -

M A19 T1

OPB-05

Fig. (69): RAPA banding patters amplifying for A. niger (A19) and its

isolate (A19 T1 10min) using primer OPB-05M (DNA marker)

= 1kb ladders.

M A19 T1

OPB-07

Fig. (70): RAPD banding patterns amplified for A. niger (A19) &

obtained isolate (A19 T1 10min) using primer OPB-07M (DNA

marker) = 100 base ladder.

- 115 -

M A19 T1

OPB-06


obtained isolate (A19 T1 10min) using OPB-06 M (DNA

marker) = 1kb ladder.

- 116 -

Table (32), sixteen positive unique marker identified mutant

A19T1 10min amplified by primer OPG-05 at

molecular weight 3458bp, primer OPA-20 at

molecular weight 211, 185bp, primer OPB-07 at

molecular weight 1601, 862, 542, 358, 263 and

193bps and primer OPH-15 at molecular weight

1456, 895, 793, 550, 414, 312 & 217 bp these sixteen

bands found only on A19T1 10min. On the other

hand, twelve unique markers for the wild strain A.

niger A19 amplified by OPB-15 primer at molecular

weight 1211bp, OPB-06 primer at molecular weight

3016 bp, OPC-10 primer at molecular weight 284 bp,

OPB-07 primer at molecular weight 1116, 738, 489,

377 and 214bp and OPH-15 primer at molecular

weight 1011, 731, 487 and 235bps.

From table (33), it can be noticed that the genetic similarity

between two genotypes wild strain A. niger A1 (potent strain) and

its mutant (A1T1 5 min) is 81% and variation is 19%, while the

genetic similarity between wild type strain of A. niger A19 (week

strain) and its mutant A19 T1 10min is 73.6% and variation 26.4%.

- 117 -

Table (32): Positive and negative unique RAPD markers and

their molecular weight for wild A. niger A19 (weak

isolate) and its obtained isolate (A19T1 10 min)

after treatment with uv irradiation.

Primers Approx. band size in bp A19 A19T1 10min

OPG-05 OPB-15 OPB-06 OPA-20

OPC-10 OPB-07

OPH-15

3458 1211 3016 211 185 284 1601 1116 862 738 542 489 377 358 263 214 193 1456 1011 895 793 731 550 487 414 312 235 217

0 1 1 0 0 1 0 1 0 1 0 1 1 0 0 1 0 0 1 0 0 1 0 1 0 0 1 0

1 0 0 1 1 0 1 0 1 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 1 1 0 1

Presence of a band in a genotype =1 Absence of the shared band in the other genotypes = 0

- 118 -

Table (33): RAPD based genetic similarity (GS) between

genotypes of A. niger: (A1 & A1 T1 5 min) - (A19 &

A19T1 10min).

Genotype A1T1 5 min A19T1 10 min

A1

A19

0.81

0.736

Gs (ij) = 2Nij/ (Ni+ Nj) (Nei and Li 1979).

Gs= genetic similarity index

Ni j = number of bands shared by i and j

Ni = number of bands present in I

Nj = number of bands present in j

Gs (A1 A1T1) = 2 NA1A1T1/ (NA1+ NA1T1)

= 2 x 30/ (39+35)

= 60/74

= 0.81

= 81.1%

Gs (A19 A19 T1) = 2 N A19 A19 T1/ (NA19 + NA19 T1)

= 2 x 39 / (51+55)

= 78/106

= 0.736

= 73.6%

GD = 1-GS

GD = genetic distance

GD A1, A1 T1 = 1-0.18 = 0.19

GDA19, A19 T1 = 1-0.736 = 0.264

- 119 -

Protein profile (SDS-PAGE) was carried out (table 34 &

Figs. 72-76) for assessing any change between A. niger isolates

(wild & mutant strains) i, e A1 & A1,T1 (5 min) - A19 & A19 T1

(10min). Twenty two bands ranged from 243.2 to 30.1kDa were

obtained in wild isolate of A. niger A1 (potent), on the other hand

after uv irradiation (5min), mutant A. niger A1 T1 5min gives

fifteen bands ranged from 235.77 to 31.96KDa.

In A. niger A19 (wild weak strain) showed twelve bands

ranged from 91.12 to 24.78KDa, while after uv irradiation

(10min) in treated isolate A19T1 10 min showed sixteen bands

ranged from 100.74 to 21.15KDa.

- 120 -

Table (34): SDS-PAGE for (A1 &A1 T1 5 min and A19 & A19 T1

10 min.).

Lane Number

Band Number

Relative Front

Mol. Wt. KDa

Peak OD

Average OD

Marker

1 1 0.195 250 99.693 60.645

1 2 0.276 130 39.525 22.645

1 3 0.352 95 42.977 25.01

1 4 0.547 72 19.883 12.309

1 5 0.655 55 14.397 7.915

1 6 0.798 36 74.681 50.151

1 7 0.958 22 72.77 50.4

A1 T1 5 min

2 1 0.202 235.773 26.977 17.367

2 2 0.234 182.171 29.821 15.674

2 3 0.338 97.873 96.187 69.053

2 4 0.361 93.214 67.105 59.868

2 5 0.383 90.436 45.358 33.877

2 6 0.451 82.363 83.451 56.348

2 7 0.497 77.386 19.315 15.613

2 8 0.55 71.736 39.961 28.765

2 9 0.595 63.963 20.646 11.268

2 10 0.637 57.418 35.529 25.226

2 11 0.671 52.263 18.735 12.452

2 12 0.745 42.049 42.864 32.169

2 13 0.769 39.302 25.163 16.795

2 14 0.817 33.973 28.233 19.969

2 15 0.838 31.964 27.821 17.347

- 121 -

Table (34): (Cont.)

Lane Number

Band Number

Relative Front

Mol. Wt. KDa

Peak OD

Average OD

A1

3 1 0.198 243.228 19.121 12.012

3 2 0.237 173.532 15.556 10.148

3 3 0.338 99.110 103.210 65.743

3 4 0.362 93.081 73.502 64.481

3 5 0.380 90.644 47.650 37.891

3 6 0.403 87.707 18.646 12.536

3 7 0.446 82.871 86.763 51.680

3 8 0.495 77.446 18.708 12.519

3 9 0.546 72.244 47.510 39.434

3 10 0.563 69.225 15.724 9.384

3 11 0.588 65.036 12.296 10.051

3 12 0.610 61.410 9.132 5.359

3 13 0.632 58.183 31.296 22.432

3 14 0.644 56.633 24.72 21.014

3 15 0.663 53.569 26.027 23.172

3 16 0.670 52.444 19.673 15.715

3 17 0.734 43.324 39.428 31.337

3 18 0.758 40.573 24.545 13.209

3 19 0.803 35.559 32.658 24.659

3 20 0.820 33.65 38.233 32.818

3 21 0.830 32.635 36.280 29.192

3 22 0.856 30.134 20.572 12.561

- 122 -

Table (34): (Cont.)

Lane Band Relative Mol. Wt. Peak Average

Number Number Front KDa OD OD

A19 T1 10min

4 1 0.334 100.74 44.953 33.827

4 2 0.36 93.352 46.105 38.476

4 3 0.375 91.469 40.654 31.026

4 4 0.439 83.677 33.673 24.112

4 5 0.451 82.311 30.023 23.159

4 6 0.54 72.987 13.304 8.649

4 7 0.593 64.262 14.782 6.193

4 8 0.635 57.587 23.222 15.373

4 9 0.662 53.746 18.875 13.385

4 10 0.72 45.318 18.272 11.959

4 11 0.743 42.854 25.743 18.948

4 12 0.765 40.693 13.331 8.78

4 13 0.808 35.854 21.529 12.773

4 14 0.819 34.505 27.362 21.497

4 15 0.839 32.25 25.599 18.881

4 16 0.973 21.15 109.716 80.623

A19 5 1 0.378 91.126 30.331 19.856

5 2 0.451 82.252 21.304 11.502

5 3 0.528 74.031 6.84 3.453

5 4 0.55 71.425 5.397 2.002

5 5 0.594 62.989 8.63 2.804

5 6 0.633 58.028 12.556 7.514

5 7 0.676 52.183 21.973 16.201

5 8 0.749 43.479 32.506 26.213

5 9 0.779 40.573 42.3 35.956

5 10 0.832 33.92 43.537 37.267

5 11 0.86 30.238 25.529 14.321

5 12 0.924 24.782 39.506 25.148

A1 : A. niger (potent wild isolate) A1 T1 : A. niger A1 after 5min uv irradiation A19 : A. niger (weak wild isolate) A19 T1 : A. niger A19 after 10min uv irradiation

- 123 -

Fig. (72): Marker Lane Profile

- 124 -

Fig. (73): Lane A1 T1 Profile.

- 125 -

Fig. (74): Lane A1 Profile.

- 126 -

Fig. (75): Lane A19 T1 Profile.

- 127 -

Fig. (76): Lane A19 Profile.

- 128 -

- 129 -

Discussion

Citric acid (a tricarboxylic acid) has a broad use in

preparation of numerous industrial products such as food,

pharmaceutical, chemicals and as a cleaning agents (Lotfy et

al., 2007; Kim et al., 2006 and Ghassempour et al., 2004). A.

niger has been the organism of choice for citric acid production

due to its ease of handling, ability to use a variety of cheep raw

material and high yield of citric acid (Torres and Garcia 2009;

Papagianni 2007; Bayraktar and Mehmetaglu, 2000; Mourya

and Jauhri, 2000; El-Holi 1999; Gutierrez-Rojas, 1995 and

Gupta and Sharma 1994).

In recent years a considerable interest has been shown in

using agriculture products and their residues as alternative sources

of carbon such for citric acid production by A. niger (Alagarsamy

and Nallusamy 2010; Rodrigues et al., 2010; Niamul, et al.,

2009; Darani and Zoghi 2008; Ali 2006; Prado et al., 2005;

Kumar et al., 2003 and Soccol 2001). These residues are very

well adapted to solid state cultures due to their cellulosic and

starchy nature, there has been an increasing trend towards

efficient utilization of these residues, besides being a form of

reducing environmental concerns (Soccol and Vandenberghe

2003).

- 130 -

In this study twenty strains of A. niger were isolated from

different sources, screened for their capacity to produce citric

acid, all the strains were able to produce citric acid in different

quantities at different time intervals i.e., 4, 8, & 12 days on

indicator medium. The best incubation period for production for

all isolates was 12 days. Citric acid productivity was obtained by

all strains under the study when using different concentration (5,

10, 15, 20 and 25%) of four carbohydrate by-products (maize

straw, potato solid wastes, sugar beet pulp and molasses) when

each used alone without any additions after 12 days incubation

and the production enhanced when the fermentation medium

amended with same concentrations of the above mentioned

substrates.

Type and concentration of carbohydrate by-product affect

the production of citric acid by A. niger strains under the study.

Sugar beet pulp & molasses giving the highest yield of citric acid,

while maize straw giving the lowest production by A. niger

strains. Production recorded when using potato wastes was more

than maize straw but lower than sugar beet pulp and molasses.

Increasing the substrate concentration under investigation led to

increase the production for all the isolates with all the

carbohydrate by-products used. Isolates (A1, A4 & A5) recorded

highest yield of citric acid production on different media used,

while A8, A16, A18, & A19 recorded weak production.

- 131 -

In view of other workers, Mehyar et al., 2005 indicated

that sugar from date extract or molasses could be used as

carbohydrate source for citric acid production, strain type, nature

of carbohydrate source and total soluble solids (TSS) has a

significant effect on citric acid production by A. niger.

Vandenberghe et al., 2004 compare citric acid production from

cassava bagasse and two other substrates (sugar cane bagasse &

coffee husk) in SSF using a culture of A. niger, cassava bagasse

giving the highest yield of citric among the tested substrates. Lu

et al., 1995 reported that kumara (Ipomoea batatas) and taro

(Colocasia esculenta) were excellent substrates for citric acid

production by solid substrates fermentation using A. niger,

conversely potato (Solanum tuberosum) was a poor substrate

although it supported profuse growth. Zafiris et al., 1994

examined three strains of A. niger for citric acid production on

orange processing wastes and found that strain NRRL 599 gave

the greatest amount of citric acid. About substrate concerning

concentrations, Mourya and Jauhri 2000 claimed that increasing

substrate concentrations (maize starch hydrolysate) gave

corresponding increase of citric acid by A. niger.

A. niger is one of the best known citric acid producers, it is

used for industrial scale production, in spite of the large number

of producer strains available, it is still important to generate

strains with advantages characteristics such as enhanced citric

- 132 -

acid production and increase rate of fermentation (Conte and

Marine 2003; Rohr et al., 1992).

Strains with superior characters such as enhanced citric

acid production are hope for investigators, the most frequently

used method for enhanced citric acid production is induction by

uv irradiation (Rodrigues et al., 2010). Since uv rays are

absorbed by Purine and Pyrimidines, making them reactive,

inducing mutations (Griffiths et al., 2006 and Zaha, 2003),

therefore mutations induced by uv can randomly provide a strain

with a higher capacity of citric acid production when compared to

the parent strain for citric acid production (Rodrigues et al.,

2010).

The most potent strains of A. niger for citric acid

production in this study obtained by isolates A1, A4 and A5 on the

different used media. These selected parental isolates were

exposed to uv light irradiation and γ-rays as a mutagenic agents as

a trail for improving their capacity for citric acid production. uv

treatment affected positively in citric acid production for treated

ones after 12 days incubation, molasses at 25% conc. only was the

best carbohydrate by product when added to the basal medium for

growing the obtained isolates after uv treatment, followed by

sugar beet pulp, while lowest production recorded with maize

straw, treated isolates growing on indicator medium and basal

- 133 -

medium with potato solid wastes almost equal, both better than

with basal medium with maize straw but lowest than basal

medium amended with molasses.

Production of parental isolates A1, A4, A5, on basal medium

amended with 25% molasses were 1.22, 1.04, 1.20 (mg/ml)

respectively after 12 days incubation while the yield ranged from

(5-5.62mg/ml) for seventeen (17) ones obtained (treated) from

parent A1 after uv irradiation (2, 5, 10, 15 & 20min) with

increases reached to 4.5 fold. As for A4 (11) eleven obtained ones

recorded 2 fold increase in production when compared to the

parental isolate (non-treated), while 9 (nine) derivatives produced

2 fold increase from A5.

Asad-ur-Rehman et al., 2003, Ikram-ul-Hag et al., 2001,

Abou-Zeid and Ashy 1984 & Khan et al.,1970 reported that

molasses a by-product of sugar industry has been a substrate of

choice for citric acid production, molasses medium as the best

fermentation medium for enhanced and consistent yield of citric

acid by A. niger strains. Ali et al., 2001; & Mattey and Allan

1990 found that the production of citric acid by A. niger is one of

the most commercially utilized examples of fungal overflow

metabolism.

Several investigators utilized uv irradiation for mutations

induction from A. niger for citric acid production. The mutant

- 134 -

strains might show several fold increase in citric acid production

as compared to wild type cultures (Lotfy et al.,2007; Ikram et

al.,2004; Asad-ur-Rehman et al.,2003; Conte and Marine

2003; Ikram et al.,2001; Rugsaseel et al.,1993 and Hamissa et

al.,1992). Lotfly et al., 2007 obtained uv mutant of A. niger in

citric acid production approximately 2-3 fold increase when

compared the parental wild type strain. UV irradiation,

ethylmethane sulfonate (EMS) and acridinc (Ao) were used to

induce citric acid over production mutants in A. niger UMIP

2564, among 15, eight of the mutant derivatives were improved

with respect to citric acid production from sucrose in batch

cultures, maximum product yield was recorded by W5 a stable uv

mutant with approximately 3.2 fold increase when compared to

wild type strain.

Ikram ul-Hag et al., 2004 subjected A. niger conidial

suspension of A. niger to uv induced mutagenesis and found that

among 3 variants, GCM was found to be a better producer of

citric acid. Mourya and Jauhri 2000 found that uv was effective

in increasing citric acid by A. niger on starch hydrolysate.

Rugsaseel et al., 1993 obtained mutants with enhanced citric acid

production from soluble starch induced from A. niger WU-2223

after uv irradiation. Hamissa et al., 1992 showed that uv

treatment resulted in the development of 31 isolate of A. niger in

citric acid production from beet molasses.

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In this study γ-rays was less effective than uv light

irradiation in increasing citric acid production when treated

selected strains A1,A4 and A5. Increase 2 fold on indicator

medium & fermentation medium amended with molasses at 25%

conc. for some derivative ones at does form 50-200Gy as

mentioned in the results, maximum production obtained after γ-

rays ranged from 2-2.4mg/ml. Positive results were obtained with

γ-rays by Alani et al., 2007 who selected mutant G4 from a strain

of A. niger after four rounds of γ-ray irradiation, the parent strain

produced 30g/kg citric acid, while the mutant produced 60g/kg

from carob under solid-state fermentation. Parvez et al., 1998

studied citric acid production from sugar cane molasses by A.

niger NIAB280 in a batch cultivation process, mutant strain

showing resistance to 2-deoxyglucose in vagals medium

containing molasses as a carbon source were induced by γ-

irradiation, among mutant strains, strain RP7 produced 1.5 fold

improvement than parental strain. Begum et al., 1990 induced

mutants of A. niger in different carbohydrate media for citric acid

fermentation by γ-rays. Golubtsova et al., 1978, 1976 & 1972

reported that the most active and resistant mutants for citric acid

production by A. niger were produced at the does of 10-50 krad

(100-500Gy) of γ-rays.

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Pronounced increase in citric acid production reached to

8.5 fold recorded in this study for derivatives ones (treated) T1

and T2 (10 min) of parental weak isolate A19 when exposed to uv

treatment as compared on indicator medium. Production of the

parental strain A19 on indicator medium was 0.60 mg/ml, it

reached to 5.35 mg/ml for A19T1 after 10 min of uv irradiation.

Mourya & Jauhri 2000 and Kunar et al., 1989 observed

variation in citric acid production of various mutants of A. niger

with sucrose as substrate, attributed this effect to the higher

glycolytic activites of these strains subsequent finding on

activities of the two glycolytic enzymes (hexokinase &

phosphofructokinase fructokinase) lent further support to this

explanation that a citric acid accumulation requires a higher

glycolotic activity. Since it is unlikely that mutations have

affected the structural genes for both the enzymes, a regulatory

gene may probably be altered, therefore it is quite possible that an

increase in glycolytic capacity of A. niger increase the ability of

these strains to accumulate citric acid.

PCR is one of the most popular tools in molecular

diagnosis. It can amplify DNA samples to a detectable signal

level within a short period of time, in theory a DNA product can

be amplified and doubled in each cycle, however, primer-diamer

and GC-rich regions of the template and the PCR system’s

heating/coaling ratio may interfere with the efficiency of the PCR

- 137 -

(Li et al., 2005 and Brownie et al., 1997). PCR amplification can

be performed directly on various microbial cultures for

filamentous fungi and yeast, prior isolation of DNA is often

preferred (Plaza et al., 2004 and Liu et al., 2000).

Data in the study revealed that RAPD analysis using PCR

successeded to differentiate between wild strain of A. niger

A1(potent isolate) and the mutant one A1 T1 (5min) after uv

irradiation-in addition to the wild strain A19 (weak isolate) and its

mutant A19 T1 (10min) after uv irradiation. Genetic similarities

between these genotypes. i.e., (A1 & A1 T1 5 min) – (A19 & A19 T1

10min) were 81.0 & 73.6% respectively, while the variation

obtained were 19.0& 26.4% respectively. Protein profile for A1

(wild potent isolate) indicated 22 bands from 243.2 to 30.1 KDa,

there were 15 bands for the obtained mutant A1 T1 5min ranged

from 235.27 to 37.96KDa. As for A19 (wild weak isolate) 12

bands were obtained ranged from 91.12 to 24.78 KDa, while 16

bands were obtained for its mutant A19 T1 (10min) ranged from

100.74 to 21.15KDa.

Mutagenic effect of uv irradiation induced these variations.

It can be noticed that variation between A19 & its mutant (26.4%)

higher than between A1 & its mutant (19%), this may explained to

the fact that citric acid production for A19 T1 10min was 9 fold

over the production of the wild isolate A19, while the production

of the mutant A1 T1 (5min) was 4 times higher than its wild A1.

- 138 -

Vossen et al., (1994) pointed to the applicability of RAPD

markers for identification purposes. The technique is based on the

amplification of random DNA sequences by polymerase chain

reaction (PCR) using arbitrary primers, they optimized the

reaction components and evaluated several primers, the RAPD

assay clearly showed comparative banding patterns for strains

within the same species. This assay enabled to discriminate

between Zygo saccharomyces bailii and rauxii. Within

Saccharomyces cerevisiae species two distinct groups of RAPD-

patterns have been found indicating the possibility to distinguish

between varieties.

- 139 -

Summary

Twenty strains of Aspergillus niger were isolated from

different sources, and screened for their capacity to produce citric

acid on indicator medium and different carbohydrate by-products

i.e. maize straw, potato solid wastes, sugar beet pulp and

molasses. The most potent strains for production were found to be

three strains, out of twenty (A1, A4 & A5) while A8, A16, A18 &

A19, were recorded weak production on indicator medium which

contain different four carbohydrate by-products under the study.

The chosen isolated isolates A1, A4 & A5 were exposed to

uv irradiation for different periods i.e., 2, 5, 10, 15, 20, 25 & 30

minutes & to different does of γ-rays (25, 50, 100 & 200)Gy as a

mutagenic agents in a trail for improving their capacity for citric

acid production, the obtained results can be summarized in the

following:-

1. Type and concentration of carbohydrate by-products under

the study affect citric acid yield by Aspergillus niger strains.

2. Increasing carbohydrate by-product concentration led to

increase the production of citric acid, the best concentration

for production was 25% for maize straw, potato solid wastes

(peels), sugar beet pulp and molasses.

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3. Citric acid productivity were obtained by all isolated strains

when using different concentration of the four carbohydrate

by-products when each used alone without any additions

after 12 days incubation and the yield enhanced when the

fermentation medium amended with the same concentrations

of the mentioned substrates.

4. Sugar beet pulp and molasses giving the highest yield by

Aspergillus niger strains under the study while maize straw

giving lowest production. The yield on indicator medium and

potato solid wastes almost equal. Both better than maize

straw but lower than sugar beet pulp and molasses.

5. UV treatment affect positively in citric acid production by

selected isolates of A. niger growing on different

carbohydrate by-products. Molasses was the best

carbohydrate by-product for citric acid production by treated

ones when added at 25% to the basal medium after 12 day

incubation followed by sugar beet pulp, while the lowest

yield recorded with maize straw. Production by treated

isolates after uv treatment growing on indicator medium and

basal medium with potato solid wastes almost equal, both

better than with maize straw but lower than molasses and

sugar beet pulp.

- 141 -

6. Production of parental isolates A1, A4 & A5 on basal medium

with 25% maize straw were: 0.73, 0.70 & 0.85(mg/ml)

respectively after 12 days incubation. Maximum yield from

the treated (obtained ones) after u.v. irradiation were: 1.50,

1.45 by A4 T2 (5min) & A4 T1 (10min) respectively with

increase 2 fold.

7. Yield of citric acid by parental isolates A1, A4, & A5, on basal

medium with 25% potato solid wastes were: 0.94, 0.91 &

0.95 (mg/ml) respectively after 12 days incubation, while

maximum yield by the treated ones after uv treatment were

1.85, 1.84 & 1.78 (mg/ml) by A1 T1 (5min), A4 T2 (5min) A1

T3 (5 min) respectively.

8. On basal medium amended with 25% sugar beet pulp, the

production of parental isolates A1, A4, & A5 after 12 day

incubation were: 1.60, 1.50 & 1.51(mg/ml) respectively after

12 days incubation, while maximum yield by the treated ones

after u.v. treatment reached to 3.16 & 2.82 (mg/ml) with

increase 2 fold by A1 T1 (5min) & A1 T5 (5min) respectively.

9. The production of citric acid on basal medium amended with

25% molasses after 12 days for the parental isolates A1, A4 &

A5 were: 1.22, 1.04 & 1.20 (mg/ml) respectively while the

yield ranged from 5-5.62mg/ml for 17 ones obtained (treated

with uv treatment) form the parental A1 after exposure times

- 142 -

2, 5, 10, 15 & 20 minutes with increase reached to 4.5 fold,

maximums yield with A1 T1 (5min) was 5.62mg/ml.

10. UV treatment was more effective than γ-rays in producing

number of obtained isolates of A. niger yielding more

production of citric acid than the parental strains. On basal

medium amended with 25% molasses, increase 2 fold in

production of citric acid for A1 after γ-rays treatment after 12

days incubation by A1T1 (100Gy), the yield reached to 2.40

(mg/ml) while it was 1.20 (mg/ml) for the wild isolate A1.

11. Increase 2 fold in citric acid production on indicator medium

for the treated A. niger after γ-rays treatment after 12 days

incubation in derivatives A1(T1 50 Gy; T1, T2, T3 & T4 100

Gy; T1 & T2 200 Gy), A4(T1, T2 & T3 100Gy), A5(T1 & T2

100 Gy; T1 & T2 200 Gy).

12. Slight increase in citric acid production in few treated ones

on basal medium with 25% maize straw when compared with

the parental isolates (untreated with γ-rays).

13. When the basal medium amended with 25% potato solid

wastes, maximum citric acid production after 12 days

incubation and γ-rays treatment were : 1.80, 1.65, 1.60 &

1.63 (mg/ml) by A1(T1, T2, T3 & T4 after exposed to 100Gy)

with increase 1.5 fold, no increase in production obtained in

- 143 -

the derivatives for A4 & A5 after all doses of γ-rays (25, 50,

100 & 200 Gy).

14. Slight increase in citric acid production for some obtained

ones on basal medium with 25% sugar beat pulp when

compared with the parental isolates (untreated with γ-rays).

15. When the basal medium amended with 25% molasses,

increase in production for ten (10) obtained ones for A1 after

treatment with γ-rays. 2 fold recorded after 12 days

incubation by A1 (T1 100 Gy) & 1.5 fold for T1 25 Gy; (T1,

T2, T3 ) 50 Gy; (T2, T3, T4) 100 Gy & (T1, T2) 200 Gy. Also

increases in production reached to 1.5 folds for some

obtained ones in this carbohydrate by-product by A4 & A5

strains.

16. Production of parental weak isolates A8, A16, A18, & A19 on

indicator medium after 12 days incubation were 0.74, 0.70,

0.69 & 0.60 (mg/ml) respectively. Interestingly, increase of

production fold reached to 5, 6, 7, 8 & 9 folds recorded after

uv irradiation. Maximum production of citric acid were:

3.60, 5.20, 5.20 & 5.35 (mg/ml) by A8T1 (10min), A16T1

(10min), A18T1 (10min) & A19T1 (10min) respectively.

17. RAPD-PCR analysis revealed that five positive unique

markers amplified by OPO-14 primer at molecular weight of

- 144 -

4245bp, OPA-18 primmer at molecular weight of 811bp, and

OPA-04 primer at molecular weight 572, 219 and 135bp

identified the mutant A1T1 5min, these five bands found only

on A1T1 5min, while in the wild isolate A1 there were nine

markers amplified by OPO-02 primer at molecular weight of

2115 and 811bp, OPO-14 primer at molecular weight pf 578

& 325bp, OPC-14 primer at molecular weight of 548bp,

OPA-05 primer at molecular weight 604 & 480bp and OPB-

05 primer at molecular weight 453 & 272bp.

18. Sixteen positive unique markers amplified by primer OPG-

05 at molecular weight 3458bp, primer OPA-20 at molecular

weight 211, 185pb, primer of OPB-07 at molecular weight

1601, 862, 542, 358, 263 and 193 bp and primer OPH-15 at

molecular weight 1456, 895, 793, 550, 414, 312, 217 bp

identified the mutant A19 T1 (10min.) while in the wild

isolate A19 twelve unique markers were found amplified by

OPB-15 primer at molecular weight 1211bp, OPB-06 primer

at molecular weight 3016bp, OPC-10 primmer at molecular

weight 284bp, OPB-07 primer at molecular weight 1116,

738, 489, 377 and 214bp1 and OPH-15 primers at molecular

weight 1011, 731, 487 and 235 bps.

19. The genetic similarity between two genotypes: wild strain A1

and its mutant A1 T1 5min is 81% and variation is 19% while

- 145 -

the genetic similarity between wild type strain of A. niger

A19 and its mutant A19 T110 min is 73.6% and variation

26.4%.

20. In protein profile (SDS-PAGE) twenty two bands ranged

from 243.2 to 30.1 KDa were obtained in A. niger (A1)

(Parental potent isolate) on the other hand after uv irradiation

(5min) mutant A. niger A1T1 (5min) have fifteen bands

ranged from 235.77 to 31.96kDa.

21. A. niger A19 (parental weak isolate) showed twelve bands

raged from 91.12 to 24.78kDa, while after uv irradiation

(10min) mutant obtained isolate A19T1 (10min) showed

sixteen bands ranged from 100.74 to 21.15kDa.

- 146 -

- 147 -

CONCLUSION

• Sugar beet pulp and molasses giving the highest yield of CA by A. niger isolates while maize straw giving the lowest CA production. The yield on synthetic medium and potato solid wastes almost equal. Both better than maize straw but lower than sugar beet pulp and molasses.

• UV treatment after positively in CA production by selected isolates of A. niger growing on different carbohydrate by-products.

- Maximum yield of CA by treated isolates of A. niger recorded increase 2 folds on basal medium amended with maize straw or sugar beet pulp with 25% conc.

- Slight increase in CA production by treated isolates of A. niger growing on basal medium amended with potato solid wastes with 25% conc.

- Maximum production of CA basal medium amended with 25% molasses by treated isolate (A1T1 5min.) reached to 4.5 folds than parental isolate (Al).

- Production of CAby weak isolate (A19T1 10 min.) reached to 8.5 folds than wild isolate (A19).

• Gamma ray was less effective than uv irradiation in increasing CA production by treated isolates. The highest production recorded increase 2 folds by treated isolates on synthetic medium and basal medium amended with molasses with 25% conc. at 100 Gy.

• The genetic similarity between two genotypes: wild strain A1 and its mutant A1T1 (5min) is 81% and variation is 19% while the genetic similarity between wild type strain of A. niger A19 and its mutant A19T1 (10 min.) is 73.6% and variation 26.4%.

- 148 -

Recommendation :

• All the isolates of A. niger have the ability to produce CA in

different quantities at different time intervals.

• The most potent isolates A1, A4 & A5 while the weak

isolates A8, A16, A18 & A19.

• The best incubation periods for CA production by isolates

was 12 days.

• Type and concentration of carbohydrate by-products affect

CA production by isolates of A. niger.

• UV treatment was more effective than γ-rays. It can

randomly provides mutant with hyperproduction for CA

when compared to the wild strain.

• UV irradiation affect positively in CA production by weak

producer of A. niger (A19T1 10 min.) more than active

producer (A1T1 5 min.).

• CA production by A1T1 5 min of A. niger on maize straw,

potato solid wastes, sugar beet pulp and molasses after 12

days incubation 1.30, 1.85, 3.16 and 5.62 mg/ml respectively

by CA production by A19T1 10 min of A. niger on the same

substrates after 12 days incubation 2.05, 2.83, 3.80 & 6.10

mg/ml respectively.

- 149 -

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- 1 -

ا�� ا��

ـ لس نيجر رجليسبة أ عزله من فطر ) ٢٠(تم عزل ادر مختلفـة ـ مـن مص ـ مقدرتهالقياس indicator)ريك علـي البيئـة الدالـة يت علي إنتاج حـامض الس

medium)قش الذرة ـ قشرة البطـاطس ـ (راتية المختلفة د والمخلفات الكربوهي ـوقد) نجر السكر ومولاس قصب السكرمخلف ب ـتزرـ أح A1, A4, A5زلات الع

هـى ,A8, A16, A18, A19هذه البيئات، بينما كانت العزلات ة علي ـأعلى إنتاجي .الأضعف إنتاجاً لذلك الحامض

للأشعة فـوق البنفسـجية A5, A4, A1عرضت العزلات الفطرية المختارة ولجرعات مختلفـة ) دقيقة ٣٠، ٢٥، ٢٠، ١٥، ١٠، ٥، ٢( لفترات زمنية مختلفة

وذلك بغرض تحسين قـدرتها علـي ) جراى٢٠٠، ١٠٠، ٥٠، ٢٥(اما من أشعة ج . إنتاج حامض السيتريك

: ملخص النتائج المتحصل عليها

، ١٥، ١٠، ٥(أثرت نوعية المخلفات الكربوهيدارتية المستخدمة وتركيزها -١علي إنتاج حامض السيتريك بواسطة عـزلات الأسـبرجلس %) ٢٥، ٢٠

.نيجر

بوهيدراتى يؤدى إلـي زيـادة إنتـاج حـامض زيادة تركيز المخلف الكر -٢هو الأمثل للأربع مخلفات المسـتخدمة % ٢٥الستيريك، حيث كان التركيز

وقد زادت إنتاجية حامض السيتريك عند إضافة نفـس التركيـزات مـن . المخلفات الكربوهيدراتية إلي البيئة الاساسية

ـ -٣ ى انتـاج جميع عزلات الاسبرجلس نيجر المختبرة كانت ذات مقدرة علحمض الستريك عند استخدام المخلفات الكربوهيدارتية الأربعة المـذكورة

- 2 -

عند استخدامها بمفردها بدون أي %) ٢٥ ، ٢٠، ١٥، ١٠، ٥(وبتركيزات . يوم تحضين١٢إضافات بعد

تم الحصول علي أعلى إنتاجية لحامض السيتريك باستخدام بنجـر السـكر -٤ بينما كان A. niger ختبرة لفطرومولاس قصب السكر لجميع العزلات الم

مخلف قش الذرة هو أقـل المصـادر الكربوهيدراتيـة المختبـرة إنتاجـاً للحامض، وكان إنتاج حامض السيتريك بواسطة العزلات المختبرة علـي بيئة البطاطس والبيئة الدالة متساوياً فكلاهما أعلى إنتاجاً من استخدام قـش

.مولاسالذرة ولكن أقل من بنجر السكر وال

أثرت المعاملة بالأشعة فوق البنفسجية إيجابياً في إنتاج حامض السـيتريك -٥بواسطة العزلات المختارة من الأسبرجلس نيجر النامية علـي المخلفـات الكربوهيدراتية الأربع حيث كان مخلف مولاس قصب السكر هو الأفضـل

ـ ة لإنتاج حامض السيتريك بواسطة عزلات الأسـبرجلس نيجـر المعاملعلي البيئة الاساسية وبعد % ٢٥بالأشعة فوق البنفسجية عند إضافته بنسبة

يوم من التحضين يليه مخلف بنجر السكر بينما كان مخلف قش الـذرة ١٢هو الأقل إنتاجية لحامض السيتريك ـ أيضاً بعد المعاملة بالأشـعة فـوق

البطـاطس كانت إنتاجية العزلات المتحصل عليها والنامية علي ةالبنفسجيفكلاهما أقل انتاجاً عـن بنجـر السـكر . والبيئة الاساسية متساوية تقريباً . والمولاس وأعلى من قش الذرة

A1, A4, A5:كانت إنتاجية حامض السيتريك للعزلات الأبوية المختـارة -٦ يـوم مـن ١٢علي التـوالي بعـد ) مل/مليجرام (٠,٨٥و٠,٧٠، ٠,٧٣

من مخلف الذرة بينما % ٢٥مضاف إليها التحضين علي البيئة الاساسية وال علـى التـوالى للعـزلات ) مـل /ملجم (١,٤٥، ١,٥٠كانت أعلى إنتاجية

- 3 -

A4T1,A4T2 دقائق من الأشعة فوق البنفسجية ١٠، ٥ عند التعرض لفترة .بزيادة قدرها ضعفين

، ٠,٩٤: A1, A4 & A5كانـت إنتاجيـة العـزلات الأبويـة المختـارة -٧يوم من التحضين علي البيئة ١٢لي التوالي بعد ع) مل/ملجم (٠,٩٥و٠,٩١

من مخلف البطاطس بينما كانـت أعلـى % ٢٥الاساسية والمضاف إليها عنـد التعـرض A1T1للعزلات ) مل/ملجم (١,٧٨، ١,٨٥، ١,٨٥إنتاجية دقائق من الأشعة فـوق ) ٥( عند التعرض لفترة A4T2دقائق ، ) ٥(لفترة

. رها مرتينالبنفسجية علي التوالي بزيادة قد

، ١,٦٠ A1, A4 & A5:كانـت إنتاجيـة العـزلات الأبويـة المختـارة -٨ يوم من التحضين علي البيئة ١٢علي التوالي بعد ) مل/ملجم(١,٥١و١,٥٠

من مخلف بنجر السكر بينما كانـت أعلـى % ٢٥الاساسية المضافة إليها عنـد A1T1بزيادة ضـعفين للعـزلات ) مل/ملجم (٢,٨٢، ٣,١٦إنتاجية

دقائق للأشـعة ) ٥(عند التعرض لفترة A1T5دقائق ، ) ٥(تعرض لفترة ال .ةفوق البنفسجي

، ١,٢٢ A1, A4 & A5:كانـت إنتاجيـة العـزلات الأبويـة المختـارة -٩يوم من التحضين علي البيئـة الاساسـية ١٢بعد ) مل/ملجم(١,٢٠و١,٠٤

من مولاس قصب السكر بينمـا كانـت الإنتاجيـة % ٢٥والمضافة إليها عزلة ١٧مل بواسطة /ملجم) ٥,٦٢ - ٥(امض السيتريك تتراوح ما بين لح

، ١٠، ٥، ٢ عند التعرض للأشعة البنفسجية لفتـرة A1متحصل عليها من مـل / ملجـم ٥,٦٢ كان أعلى إنتـاج - ضعف ٤,٥ دقيقة بزيادة ٢٠، ١٥

.دقائق) ٥( عند التعرض لفترة A1 T1بواسطة

أفضل عن المعاملـة بأشـعة جامـا المعاملة بالأشعة فوق البنفسجية كانت -١٠مل / ملجم ٢,٤٠وكانت أفضل النتائج المتحصل عليها من هذه المعاملة هي

- 4 -

يوم من التحضين علي البيئة الدالة المضـاف إليهـا ١٢بزيادة مرتين بعد عند تعرضـها لأشـعة جامـا A1من مولاس قصب السكر للعزلة % ٢٥

مل /ملجم١,٢٠ريك حوالي جراي بينما كان إنتاج حامض السيت ١٠٠بمقدار . الأبويةA1للعزلة

– A1بزيادة انتاجية حامض السيتريك مرتين للعزلات المتحصل عليها من -١١ يوم من التحضين على البيئة الدالة عنـد تعرضـها ١٢العزلة الأبوية بعد عنـد A4 جراى ومن العزلة الابوية ٢٠٠، ١٠٠، ٥٠لأشعة جاما بمقدار

عنـد A5 جراى وعند العزلة الابويـة ١٠٠تعرضها لاشعة جاما بمقدار . جراى٢٠٠ و ١٠٠تعرضها لاشعة جاما بمقدار

أدت المعاملة بأشعة جاما إلى زيادة إنتاجية حامض السـيتريك للعـزلات -١٢ يـوم مـن ١٢ زيادة ضـعيفة بعـد A1, A4 & A5المتحصل عليها من

من مخلف قش الـذرة % ٢٥ضاف إليها التحضين على البيئة الأساسية الم . بالمقارنة بالعزلات الأبوية

زيادة إنتاجية حامض السيتريك مرة ونصف للعزلات المتحصل عليها مـن -١٣A1 يوم من التحضين على البيئة الأساسية المضاف ١٢ العزلة الأبوية بعد

و ١,٦٠، ١,٦٥، ١,٨٠من مخلف البطاطس بزيـاده قـدرها % ٢٥إليها عند تعرضها لأشعة جاما A1(T1, T2, T3 & T4)مل بواسطة / ملجم١,٦٣

جراى بينما لا توجد زيادة لأنتاجية حامض السيتريك للعزلات ١٠٠يمقدار ، ٢٥ عند تعرضها لأشعة جاما بالجرعات A5 و A4المتحصل عليها من

. جراى٢٠٠ و ١٠٠، ٥٠

ك للعـزلات أدت المعاملة بأشعة جاما إلى زيادة إنتاجية حامض السـيتري -١٤ يـوم مـن ١٢ زياده ضـعيفه بعـد A1, A4 & A5المتحصل عليها من

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من مخلف بنجر السكر % ٢٥التحضين على البيئة الأساسية المضاف إليها . بالمقارنة بالعزلات الأبوية

زيادة إنتاجية حامض السيتريك لعشر عزلات متحصل عليها مـن العزلـة -١٥البيئة الأساسية المضاف إليهـا يوم من التحضين على ١٢ بعد A1الأبوية

من المولاس زياده قدرها مرتين عند تعرضها لأشعة جاما بمقـدار % ٢٥ جراى وزيادة قدرها مره ونصف عند تعرضها لأشعة جاما بمقـدار ١٠٠أيضاً زيادة قدرها مره ونصف بواسـطة . جراى ٢٠٠ و ١٠٠، ٥٠، ٢٥

. على نفس البيئةA5 & A4العزلات المتحصل عليها من

لحمـض A8, A16 , A18 & A19كانت إنتاجية العزلات الأبوية الضعيفة -١٦ ١٢علي الترتيب بعد ) مل/ملجم (٠,٦٠ و ٠,٦٩، ٠,٧٠، ٠,٧٤ :السيتريك

يوم تحضين ، بعد المعاملة بالأشعة فوق البنفسجية كانت أعلـى إنتاجيـة ) مـل /ملجـم (٥,٣٥ و ٥,٢، ٥,٢، ٣,٦للعزلات المتحصل عليهـا هـى

-A دقائق ،١٠ عند A16 T1 دقائق، ١٠ عند A8 T1 على التوالى للعزلات

18 T1 دقائق و١٠ عند A19T1 دقائق علي التوالي وبزيادة قـدرها ١٠ عند . مرات٨، ٧، ٦، ٥

استخدمت طريقة من طرق البصمة الوراثية وهي طريقة التكبير العشوائي -١٧يـب لتوصيف التباين بـين التراك ) RAPDs(لقطع من الحامض النووي

) uvللسلالة الأم والسلالة المتحصل عليها بعد المعاملة بأشعة الـ (الوراثية بادئ عشوائي للتقييم الـوراثي ) ١٠(وقد استخدم . على المستوى الجزيئي

DNAعلى المستوى الجزيئ للتكبير العشوائي لقطع من الحامض النووي A1T1 (5 min (وتشير النتائج إلى وجود خمس دلائل إيجابية فريدة للطفرة

OPA-18 ، البادئ ٥٤٢٤عند الوزن الجزئي OPO-14بواسطة البادئ

، ٥٧٢ عند الـوزن الجزئـي OPA-04، البادئ ٨١١عند الوزن الجزئي

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وهذه الخمس حلقات موجودة بهذه السلالة فقط بينمـا العزلـة ١٣٥، ٢١٩عنـد OPO-02فهناك تسعة دلائل فريدة بواسطة البـادئ ) الأصلية(الأم

، ٥٧٣ عند الوزن الجزيئ OPO-14، البادئ ٨١١، ٢١١٥لوزن الجزئي ا عند OPA-05، البادئ ٥٤٨عند الوزن الجزيء OPC-14، البادئ ٣٢٥

، ٤٥٣عند الوزن الجزئـي OPB-05 والبادئ ٤٨٠، ٦٠٤الوزن الجزئ ٢٧٢.

بواسـطة A19 T1 (10min) هناك ستة عشر دلائل إيجابية فريدة للطفرة -١٨عنـد OPA-20، البـادئ ٣٤٥٨عند الوزن الجزئي OPG-05البــادئ

، ٨٦٢، ١٦٠١ عند الوزن الجزئي OPB-07، ١٨٥، ٢١١الوزن الجزئي ، ١٤٥٦عند الوزن الجزئي OPH-15 والبادئ ١٩٣، ٢٦٣، ٣٥٨، ٥٤٢ بينما هناك أثني عشر دلائل فريدة ٢١٧، ٣١٢، ٤١٤، ٥٥٠، ٧٩٣، ٨٩٥

عنـد الـوزن OPB-15دئ بواسطـــة البـا A19للعزلة الفطرية الأم والبـادئ ٣٠١٦ عند الـوزن الجزئـي OPB-06 البادئ ١٢١١الجزئي

OPC-10 البادئ ٢٨٤عند الوزن الجزئي ،OPB-07 عند الوزن الجزئي عند الوزن الجزئي OPA-15، والبادئ ٢١٤، ٣٧٧، ٤٨٩، ٧٣٨، ١١١٦٢٣٥، ٤٨٧، ٧٣١، ١٠١١bp.

الأكثر إنتاجـاً لحـامض ( A1م كانت نسبة التشابه الوراثي بين العزلة الأ -١٩الستريك لفطر الأسبرجلس نيجر والطفرة المتحصل عليها بعـد التعـرض

بينما نسبة % ١٩ونسبة الاختلاف % ٨١ A1 T1 دقائق ٥ لمدة uvلأشعة والطفـرة ) الأقل إنتاجـاً ( لفطر الأسبرجلس نيجر A19التشابه للعزلة الام

A19 مدة عشـرة دقائــق بuvالمتحصل عليها بعد التعرض لأشعة

T1٢٦,٤ونسبة الاختلاف % ٧٣,٦.%

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أثنين وعشرين حزمـة (SDS-PAGE)اظهر الفصل الكهربي للبروتين -٢٠بينما هناك خمسـة ) ٣٠,١KDa – ٢٤٣,٢( بين A1للعزلة الأم ) باندات(

– ٢٣٥,٧٧ بــين A1 T1 (5min)للطفــرة ) بانــدات(عشــر حزمــة ٣١,٩٦KDa .

-٩١,١٢بـين ) بانـدات (أثني عشـر حزمـة A19أظهرت العزلة الأم -٢١٢٤,٧٨KDa للطفـرة ) بانـدات ( بينما هناك ستة عشر حزمـةA19 T1

(10min) ٢١,١٥ -١٠٠,٧٤ بينkDa .

a thesisa thesis - iaea

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